TW202444906A - Compositions and methods for the targeting of pcsk9 - Google Patents

Abstract

Provided herein are gene repressor systems comprising fusion proteins, such as fusion proteins comprising catalytically-dead Class 2, Type V CRISPR proteins, and guide nucleic acids (gRNA) useful in the repression of a proprotein convertase subtilisin/kexin Type 9 (PCSK9) gene. Also provided are methods of making, and methods of using such systems to repress transcription of PCSK9.

Description

Compositions and methods for targeting PCSK9

In mammals, cholesterol is transported by emulsification within lipoproteins. Lipoprotein particles are classified based on their density: low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), high-density lipoprotein (HDL), and chylomicrons. Surface LDL receptors are internalized during cholesterol absorption. Cells with sufficient cholesterol will block their LDL receptor synthesis to prevent new cholesterol absorption in LDL particles. Conversely, when cells are cholesterol-deficient, LDL receptor synthesis is promoted. When this process is unregulated, excess LDL particles will travel in the blood without being taken up by LDL receptors. LDL particles in the blood are oxidized and taken up by macrophages, which then become congested and form foam cells. These foam cells can become trapped in blood vessel walls and contribute to the formation of atherosclerotic plaques, one of the leading causes of heart attacks, strokes and other serious medical problems.

The hepatic protein proprotein convertase subtilisin/kexin Type 9 (PCSK9) is a secreted, globular, autoactivating serine protease that binds to the low-density lipoprotein receptor (LDL-R) during endocytosis of LDL particles, preventing LDL-R recycling to the cell surface, resulting in decreased clearance of LDL-cholesterol. PCSK9 binds to the LDL-R (via the EGF-A domain), preventing conformational changes in the receptor-ligand complex and instead redirecting the LDL-R to the lysosome. Because receptors for low-density lipoprotein particles (LDL) normally transport thousands of fat molecules (including cholesterol) per particle in the extracellular fluid, blocking or inhibiting the function of PCSK9 to enhance LDL-R-mediated clearance of LDL cholesterol can reduce LDL particle concentrations. PCSK9 is primarily expressed in the liver, intestines, kidneys, and central nervous system, but is also highly expressed in arterial walls (such as endothelial smooth muscle cells and macrophages), with local effects that can regulate vascular homeostasis and atherosclerosis.

PCSK9 is a member of the proprotein convertase (PC) family and is genetically mutated in approximately 2% to 3% of individuals with familial hypercholesterolemia (FH) (Sepideh Mikaeeli, S. et al., Functional analysis of natural PCSK9 mutants in modern and archaic humans. FEBS J. 2019 Aug 6. doi: 10.1111/febs.15036). Researchers have identified several PCSK9 mutations that cause hereditary high cholesterol (hypercholesterolemia). These mutations change a single amino acid in the PCSK9 protein. Researchers describe mutations that cause hypercholesterolemia as "gain-of-function" because they appear to enhance the activity of the PCSK9 protein or give the protein a new, non-canonical function (Blesa, S. et al., A New PCSK9 Gene Promoter Variant Affects Gene Expression and Causes Autosomal Dominant Hypercholesterolemia. J. Clin. Endocrinol. & Metab. 93:3577 (2008)). Overactive PCSK9 protein substantially reduces the number of low-density lipoprotein receptors on the surface of liver cells. People with gain-of-function mutations in the PCSK9 gene have extremely high cholesterol levels in their blood because there are fewer receptors to remove low-density lipoprotein from the blood. Autosomal dominant hypercholesterolemia (ADH) is a genetic disorder characterized by elevated low-density lipoprotein (LDL)-cholesterol levels, leading to a high risk of premature cardiovascular disease. Approximately 10 mutations in PCSK9 have been identified as the cause of the disease in different populations. All known mutations in PCSK9 that cause hypercholesterolemia result in increased enzymatic activity of this protease (Bleasa, S., 2008). In addition, mutations in PCSK9 may cause autosomal dominant familial hypo-β-lipoproteinemia, which may lead to hepatic steatosis, cirrhosis, and other symptoms.

The advent of CRISPR/Cas systems and the programmable nature of these minimal systems facilitate their use as a general technology for genome manipulation and engineering. However, current methods for generating PCSK9 protective variants and loss-of-function mutants in vivo have been ineffective due to the large number of cells that need to be modified to regulate cholesterol levels. Other issues involve off-target effects that may result from genome editing, genome instability or oncogenic modifications, and the lack of safe delivery modes for suppressor protein systems. Additionally, in certain disease indications, gene silencing or suppression is preferred over gene editing. The ability to render CRISPR nucleases such as Cas9 and CasX catalytically inactive has been demonstrated (WO2020247882A1 and US20200087641A1, incorporated herein by reference), making these systems an attractive platform for generating fusion proteins with repressor domains capable of silencing genes. Although certain repressor systems have been described, there remains a need for additional gene repressor systems that have been optimized and/or provide improvements over earlier generations of gene repressor systems, such as those based on Cas9 for use in various therapeutic, diagnostic, and research applications. Thus, there remains a need for improved compositions and methods for modulating PCSK9.

The present invention provides systems comprising or encoding fusion proteins comprising a DNA binding domain and a linked inhibitory subdomain for inhibition and/or epigenetic modification of a target nucleic acid sequence of a proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) gene. In some aspects, the fusion protein comprises: a DNA binding protein comprising a catalytically inactive CRISPR protein, such as a class 2 type V CRISPR protein; and a guide nucleic acid comprising a targeting sequence complementary to the target nucleic acid sequence of the PCSK9 gene. The protein and guide nucleic acid can be modified to passively enter the target cell and are suitable for a variety of methods for inhibiting PCSK9 , which are also provided. The present disclosure also provides vectors and lipid nanoparticles (LNPs) encoding or encapsulating the fusion protein and guide nucleic acid components to deliver the system to cells for transcriptional inhibition of the PCSK9 target nucleic acid sequence. The present disclosure also provides methods for modifying target nucleic acid sequences of the PCSK9 gene. The present disclosure also provides methods for treating individuals suffering from PCSK9-related diseases. In some embodiments, the compositions and methods have utility in individuals suffering from metabolic disorders such as (but not limited to) familial hypercholesterolemia, familial hypo-β-lipoproteinemia, or elevated cholesterol levels.

In other aspects, provided herein are systems comprising a PCSK9 inhibitory system, or vectors comprising or encoding a PCSK9 inhibitory system, for use in the manufacture of a medicament for treating a PCSK9-related disease in an individual in need thereof.

Other features and advantages of certain embodiments of the present invention will become more apparent from the following description of the embodiments and the drawings thereof and from the scope of the claims.

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The contents of WO 2020/247882, WO 2020/247883, WO 2021/113772, WO 2022/120095, WO 2022/125843, WO 2022/261150, WO 2022/261149, WO 2023/049872, WO 2023/049742 and WO 2023/240162, which disclose CasX variants and gRNA variants and methods of delivery thereof, and the contents of WO 2021/142342, which discloses compositions and methods for modifying PCSK9, are hereby incorporated by reference in their entirety.

Cross-references of related applications

This application claims priority to and the benefits of U.S. Provisional Application No. 63/492,978 filed on March 29, 2023 and U.S. Provisional Application No. 63/505,888 filed on June 2, 2023, the contents of each of which are incorporated herein by reference in their entirety. Citation of Electronic Sequence Listing

The contents of the electronic sequence listing (SCRB_058_02TW_SeqList_ST26.xml; size: 141,865,746 bytes; and creation date: March 22, 2024) are incorporated herein by reference in their entirety.

Although exemplary embodiments have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will recognize numerous variations, changes, and substitutions without departing from the spirit of the invention. Therefore, it should be understood that various alternatives to the embodiments specifically described herein may be used to practice the embodiments disclosed herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the invention, suitable methods and materials are described below. In the event of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Those skilled in the art will now contemplate numerous variations, changes, and substitutions without departing from the spirit of the present disclosure and the scope of the patent application. Definitions

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a host cell" includes two or more such host cells, reference to "a dCasX protein" includes one or more dCasX proteins, reference to "a nucleic acid sequence" includes one or more nucleic acid sequences, and the like.

As used herein, the term "about" is understood by one of ordinary skill in the art and may vary to some extent depending on the context in which it is used. If a term is used that is not clear to one of ordinary skill in the art, given the context in which the term "about" is used, "about" will mean up to plus or minus 10% of the particular term.

Those skilled in the art will understand that, for any and all purposes, all ranges disclosed herein also include any and all possible subranges and combinations of subranges thereof. In addition, those skilled in the art will understand that a range includes each individual member. Thus, for example, a group having 1 to 3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 1 to 5 members refers to groups having 1, 2, 3, 4, or 5 members, and so on.

The term "combinations thereof" includes every possible combination of the elements referred to by the term.

As used herein, the term "exemplary" refers to an example or illustration, and is not intended to imply any preference or value.

As used herein, the term "CasX protein" refers to a family of proteins, including, for example, all naturally occurring CasX proteins ("reference CasX"), as well as CasX proteins with sequence modifications, such as dCasX, which have one or more improved features relative to the CasX protein from which they are derived, as described more fully below.

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to polymeric forms of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, the terms "polynucleotide" and "nucleic acid" encompass single-stranded DNA; double-stranded DNA; multiple-stranded DNA; single-stranded RNA; double-stranded RNA; multiple-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

"Hybridable" or "complementary" are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) comprises a nucleotide sequence that enables it to non-covalently bind (i.e., form Watson-Crick base pairs and/or G/U base pairs), "attach" or "hybridize" with another nucleic acid in a sequence-specific, antiparallel manner (i.e., nucleic acid-specific binding to complementary nucleic acids) under appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. It is understood that a polynucleotide sequence need not be 100% complementary to a target nucleic acid with which it can specifically hybridize; it can have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity with a target nucleic acid and still hybridize with the target nucleic acid. In addition, polynucleotides may be hybridized in one or more segments such that intermediate or adjacent segments do not participate in the hybridization event (e.g., loop structures or hairpin structures, "bulges," "bubbles," and the like). Thus, one skilled in the art will appreciate that while individual bases within a sequence may not be complementary to another sequence, the sequences as a whole are still considered complementary.

For purposes of the present invention, "gene" includes DNA regions that encode a gene product (e.g., protein, RNA), as well as all DNA regions that regulate the production of a gene product, whether or not such regulatory sequences are adjacent to the coding sequence and/or the transcribed sequence. Thus, a gene may include accessory element sequences, which include, but are not necessarily limited to, promoter sequences, terminators, translational regulatory sequences (such as ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions. Coding sequences encode gene products after transcription or after transcription and translation; the coding sequences of the present disclosure may comprise fragments and may not necessarily contain a full-length open reading frame. A gene may include both transcribed strands and complementary strands containing anticodons.

The term "downstream" refers to a nucleotide sequence located 3' of a reference nucleotide sequence. In certain embodiments, a downstream nucleotide sequence is a sequence after the transcription start site. For example, the transcription start codon of a gene is located downstream of the transcription start site.

The term "upstream" refers to a nucleotide sequence located 5' of a reference nucleotide sequence. In certain embodiments, an upstream nucleotide sequence is associated with a sequence located 5' to a coding region or a transcription start site. For example, most promoters are located upstream of a transcription start site.

The term "adjacent" with respect to polynucleotide or amino acid sequences refers to sequences that are close to or adjacent to each other in a polynucleotide or polypeptide. A skilled artisan will appreciate that two sequences can be considered adjacent to each other and still encompass a limited number of intervening sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides or amino acids.

The term "regulatory element" is used interchangeably herein with the term "regulatory sequence" and is intended to include promoters, enhancers, and other expression regulatory elements. It will be understood that the selection of appropriate regulatory elements will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid contains multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

The term "auxiliary element" is used interchangeably herein with the term "auxiliary sequence" and is intended to include coding and non-coding sequences that enhance nucleic acid expression, nucleic acid transport, or mRNA or protein function, and particularly includes polyadenylation signals (poly(A) signals), enhancer elements, introns, post-transcriptional regulatory elements (PTREs), nuclear localization signals (NLSs), deaminases, DNA glycosidase inhibitors, additional promoters, factors that stimulate CRISPR-mediated homology-directed repair (e.g., in cis or trans), self-cleavage sequences, and fusion domains (e.g., fusion domains fused to a CRISPR protein). It will be understood that the selection of the appropriate one or more auxiliary elements will depend on the coding component to be expressed (e.g., protein or RNA) or on whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

The term "promoter" refers to a DNA sequence containing a transcription start site and additional sequences that promote polymerase binding and transcription. Exemplary eukaryotic promoters include elements such as TATA boxes and/or B recognition elements (BREs), and assist or promote the transcription and expression of related transcribable polynucleotide sequences and/or genes (or transgenic genes). Promoters can be produced synthetically or can be derived from a known or naturally occurring promoter sequence or another promoter sequence. The promoter can be proximal or distal to the gene to be transcribed. Promoters can also include chimeric promoters, which include a combination of two or more heterologous sequences to impart certain properties. The promoter disclosed herein may include variants of promoter sequences that are similar in composition to other promoter sequences known or provided herein, but not identical thereto. Promoters can be classified according to criteria related to the expression pattern of the associated coding or transcribable sequence or gene operably linked to the promoter (such as constitutive, developmental, tissue-specific, inducible genes, etc.). Promoters can also be classified according to their strength. As used in the context of promoters, "strength" refers to the transcription rate of the gene controlled by the promoter. A "strong" promoter means a higher transcription rate, while a "weak" promoter means a relatively low transcription rate.

The promoter of the present invention may be a polymerase II (Pol II) promoter. Polymerase II transcribes all protein coding and many non-coding genes. Representative Pol II promoters include a core promoter, which is a sequence of about 100 base pairs surrounding the transcription start site and serves as a binding platform for the Pol II polymerase and related universal transcription factors. A promoter may contain one or more core promoter elements, such as a TATA box, a BRE, an initiator (INR), a motif ten element (MTE), a downstream core promoter element (DPE), a downstream core element (DCE), although core promoters lacking such elements are known in the art. All Pol III promoters are contemplated to be within the scope of the present disclosure.

The promoter of the present disclosure may be a polymerase III (Pol III) promoter. Pol III transcribes DNA to synthesize small ribosomal RNAs, such as 5S rRNA, tRNA, and other small RNAs. Representative Pol III promoters use internal control sequences (sequences within the transcribed segment of a gene) to support transcription, but sometimes upstream elements such as a TATA box are also used. All Pol III promoters are contemplated to be within the scope of the present disclosure.

The term "enhancer" refers to a regulatory DNA sequence that, when bound to specific proteins called transcription factors, regulates the expression of an associated gene. An enhancer may be located in an intron of a gene, or 5' or 3' to the gene coding sequence. An enhancer may be proximal to a gene (i.e., within tens or hundreds of base pairs (bp) of the promoter), or may be distal to a gene (i.e., thousands, hundreds of thousands, or even millions of bp from the promoter). A single gene may be regulated by more than one enhancer, and it is contemplated that such more than one enhancer is within the scope of the present disclosure.

As used herein, "post-transcriptional regulatory element (PTRE)", such as hepatitis PTRE, refers to a DNA sequence that, when transcribed, generates a tertiary structure capable of exhibiting post-transcriptional activity to enhance or promote the expression of an associated gene to which it is operably linked.

"Operably linked" means the joining of two or more components (such as sequence elements) wherein the components are arranged so that the two components function normally and at least one of the components can mediate a function imposed on at least one of the other components (such as a promoter and a coding sequence). Those skilled in the art will appreciate that two components need not be physically connected to be operably linked.

In the context of the present invention and with respect to genes, "repress", "repression", "repressing", "transcriptional repression", "inhibition of gene expression", "downregulation" and "silencing" are used interchangeably herein to refer to the inhibition or blocking of transcription of a gene or a portion thereof. Thus, repression of a gene can result in a reduction in the production of the gene product. Examples of gene repression processes that reduce transcription include, but are not limited to, processes that inhibit the formation of a transcription initiation complex, processes that reduce the rate of transcription initiation, processes that reduce the rate of transcription elongation, processes that reduce transcription persistence, and processes that antagonize transcriptional activation (e.g., by blocking the binding of a transcriptional activating factor). Gene repression can constitute, for example, prevention of activation as well as inhibition of expression below the existing amount. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription; the latter can be caused by epigenetic modifications of the gene.

"Repressor" or "repressor domain" are used interchangeably to refer to a polypeptide factor that acts as a regulatory element on DNA that represses, inhibits or blocks DNA transcription, resulting in inhibition of gene expression. In the context of the present invention, the association of a repressor domain with a DNA binding protein can prevent auto-promoter transcription or otherwise repress gene expression when bound to a target nucleic acid. Without wishing to be bound by theory, it is believed that transcriptional repressors may act by a variety of mechanisms, including physically blocking RNA polymerase access by steric hindrance, altering the post-translational modification state of the polymerase, modifying the epigenetic state of nascent RNA, altering the epigenetic state of DNA via methylation, altering the epigenetic state of DNA via histone deacetylation or regulation of nucleosome remodeling, or preventing enhancer-promoter interactions, thereby causing gene silencing or reduced gene expression.

"Long-term suppressor fusion protein" or "LTRP" is used interchangeably herein with "suppressor fusion protein" and refers to a fusion protein comprising a DNA binding protein (or a DNA binding domain of a protein) fused to one or more domains that is capable of inhibiting the transcription of a target nucleic acid sequence. Optionally, the long-term suppressor fusion proteins of the present disclosure may contain additional elements, such as linkers between any of the domains of the fusion protein, nuclear localization signals, nuclear export signals, and additional protein domains that confer additional activities to the long-term suppressor fusion protein.

As used herein, the "LTRP:gRNA system" is a system for transcriptional repression and comprises: a long-term repressor fusion protein comprising a catalytically inactive CRISPR protein and one or more linked repressor domains; and a guide nucleic acid (gRNA) that binds to the catalytically inactive CRISPR protein. For clarity, the system also includes any encoding DNA, RNA or vector and the like that can be used to generate the long-term repressor fusion protein and gRNA components of the system.

As used herein, a "catalytically inactive DNA binding protein" refers to a protein or protein domain that is able to bind DNA but is unable to cut or cleave DNA. As used herein, a "catalytically inactive CRISPR protein" refers to a CRISPR protein that lacks endonuclease activity. Those skilled in the art will appreciate that a CRISPR protein can be catalytically inactive and still able to perform additional protein functions, such as DNA binding. Similarly, a "catalytically inactive CasX" refers to a CasX protein that lacks endonuclease activity but is still able to perform additional protein functions (such as DNA binding).

As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of selection, restriction and/or ligation steps, resulting in a construct having a structural coding or non-coding sequence that is distinguishable from endogenous nucleic acids found in natural systems. In general, DNA sequences encoding structural coding sequences can be assembled from cDNA fragments and short oligonucleotide linkers or from a series of synthetic oligonucleotides to provide synthetic nucleic acids that can be expressed from recombinant transcriptional units contained in cells or in acellular transcription and translation systems. These sequences can be provided in the form of open reading frames that are not interrupted by internal non-translated sequences or introns (which are typically present in eukaryotic genes). Genomic DNA containing the relevant sequence can also be used to form recombinant genes or transcriptional units. Non-translated DNA sequences may be present 5' or 3' to the open reading frame, wherein these sequences do not interfere with manipulation or expression of the coding region and may in fact be used to regulate the production of the desired product by a variety of mechanisms (see "enhancer" and "promoter" above).

The term "recombinant polynucleotide" or "recombinant nucleic acid" refers to a non-natural existence, for example, produced by artificially combining two otherwise separate segments of a sequence through human intervention. This artificial combination is usually achieved by chemical synthesis means or by artificial manipulation of separate segments of nucleic acids, such as by genetic engineering techniques. Such manipulations are usually performed to replace codons with coding codons or conserved amino acids, while typically introducing or removing redundant codons at sequence recognition sites. Alternatively, the manipulation is performed to connect nucleic acid segments with desired functions together to produce a desired functional combination. This artificial combination is usually achieved by chemical synthesis means or by artificial manipulation of separate segments of nucleic acids, such as by genetic engineering techniques.

Similarly, the term "recombinant polypeptide" or "recombinant protein" refers to a polypeptide or protein that does not occur in nature, for example, by artificially combining two otherwise separate segments of amino acid sequence through human intervention. Thus, for example, a protein comprising a heterologous amino acid sequence is recombinant.

As used herein, "lipid nanoparticles" or "LNPs" refer to particles having at least one nanometer dimension (e.g., 1 to 1,000 nm) that include one or more lipids (e.g., cationic lipids, non-cationic lipids, co-phospholipids, and PEG-modified lipids) and cholesterol. Specific components of LNPs are described more fully below. Lipid nanoparticles can be included in formulations that can be used to deliver active agents or therapeutic agents, such as nucleic acids (e.g., mRNA) to target sites of interest (e.g., cells, tissues, organs, tumors, and the like). The lipid nanoparticles of the present disclosure may include nucleic acids. Such lipid nanoparticles typically include neutral lipids, charged lipids, steroids, and polymer-bound lipids. Active agents or therapeutic agents, such as nucleic acids, can be encapsulated in the lipid portion of the lipid nanoparticles or in the aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticles, thereby protecting them from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, such as adverse immune responses.

As used herein, "lipid encapsulated" refers to providing an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA, gRNA, or both), to a lipid nanoparticle that is fully encapsulated, partially encapsulated, or both. The nucleic acid (e.g., mRNA) can be fully encapsulated in the lipid nanoparticle.

As used herein, "lipoproteins" (such as VLDL, LDL, and HDL) refer to a group of proteins found in serum, plasma, and lymph and are important for lipid transport. The chemical composition of each lipoprotein differs, for example, because HDL has a higher ratio of protein to lipid, while VLDL has a lower ratio of protein to lipid.

As used herein, "atherosclerosis" means hardening of the arteries that affects large and medium-sized arteries and is characterized by the presence of fatty deposits. The fatty deposits are called "atheromas" or "plaques," which are composed primarily of cholesterol and other fats, calcium, and scar tissue, and destroy the lining of the arteries.

As used herein, "coronary heart disease (CHD)" means the narrowing of the small blood vessels that supply blood and oxygen to the heart, usually caused by atherosclerosis.

As used herein, "dyslipidemia" refers to a lipid and/or lipoprotein metabolism disorder, including lipid and/or lipoprotein overproduction or deficiency. Dyslipidemia can be manifested as an increase in lipids (such as chylomicrons, cholesterol and triglycerides) and lipoproteins (such as low-density lipoprotein (LDL) cholesterol).

As used herein, "high-density lipoprotein-C" or "HDL-C" means cholesterol associated with high-density lipoprotein particles. The concentration of HDL-C in serum (or plasma) is usually quantified in mg/dL or nmol/L. "Serum HDL-C" and "plasma HDL-C" mean HDL-C in serum and plasma, respectively.

As used herein, "low-density lipoprotein-cholesterol (LDL-C)" means cholesterol carried in low-density lipoprotein particles. The concentration of LDL-C in serum (or plasma) is usually quantified in mg/dL or nmol/L. "Serum LDL-C" and "plasma LDL-C" mean LDL-C in serum and plasma, respectively.

As used herein, "hypercholesterolemia" means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol, and VLDL-cholesterol consistent with the National Cholesterol Education Program (NCEP) Panel Report Guidelines for Detection, Assessment, and Treatment of Hypercholesterolemia in Adults (see, Arch. Int. Med. 148: 36 (1988)).

As used herein, "hyperlipidemia" or "hyperlipemia" is a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition manifests itself in abnormally high concentrations of fats. The lipid fractions in the circulating blood are cholesterol, low-density lipoproteins, very low-density lipoproteins, chylomicrons, and triglycerides. The Fredrickson classification of hyperlipidemia is based on the pattern of TG and cholesterol-rich lipoprotein particles measured by electrophoresis or ultracentrifugation, and is often used to characterize the primary causes of hyperlipidemias such as hypertriglyceridemia.

As used herein, "triglyceride" or "TG" means a lipid or neutral fat composed of glycerol and three fatty acid molecules.

As used herein, "hypertriglyceridemia" means a condition characterized by elevated triglyceride levels. Its causes include primary (i.e., genetic) and secondary (other potential causes, such as diabetes, metabolic syndrome/insulin resistance, obesity, lack of exercise, smoking, excessive alcohol consumption, and a very high carbohydrate diet) factors, or most commonly a combination of both.

As used herein, "diabetes mellitus" or "diabetes" is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia), which is caused by insufficient insulin levels or decreased insulin sensitivity. Characteristic symptoms are excessive urine production (polyuria) due to high blood sugar levels; excessive thirst and increased fluid intake (polydipsia) due to increased urination to compensate for the loss of urine; blurred vision due to the effects of high blood sugar on eye optics; unexplained weight loss; and lethargy.

As used herein, "diabetic dyslipidemia" or "type 2 diabetes with dyslipidemia" means a condition characterized by type 2 diabetes, low HDL-C, elevated triglycerides (TG), and elevated small dense LDL particles.

As used herein, the term "contact" means to establish a physical connection between two or more entities. For example, contacting a target nucleic acid with a guide nucleic acid means that the target nucleic acid and the guide nucleic acid share a physical connection; for example, if the sequences share sequence similarity, they can hybridize.

"Dissociation constant" or " _Kd " is used interchangeably and refers to the affinity between a ligand "L" and a protein "P"; that is, how tightly a ligand binds to a particular protein. It can be calculated using the formula _Kd = [L][P]/[LP], where [P], [L], and [LP] represent the molar concentrations of the protein, ligand, and complex, respectively.

As used herein, the term "knock-down" refers to a reduction in the expression of a gene or its gene product. As a result of gene knockdown, protein activity or function may be attenuated or protein levels may be reduced or eliminated.

By a polynucleotide or polypeptide having a certain percentage of "sequence similarity" or "sequence identity" to another polynucleotide or polypeptide is meant the percentage of bases or amino acids that are identical when aligned and that are in the same relative position when the two sequences are compared. Sequence similarity (sometimes referred to as percentage similarity, percentage identity, or homology) can be determined in a number of different ways. To determine sequence similarity, sequences can be aligned using methods and computer programs known in the art, including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov/BLAST. The percentage of complementarity between specific stretches of nucleic acid sequences within a nucleic acid can be determined using any convenient method. Exemplary methods include the BLAST program (Basic Local Alignment Search Tool and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Unix version 8, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

The terms "polypeptide" and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coding and non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences.

A "vector" or "expression vector" is a replicon, such as a plasmid, bacteriophage, virus or cosmid, that can include another DNA segment, the expression cassette, to cause the replication or expression of the other DNA segment in the cell.

The term "naturally-occurring" or "unmodified" or "wild type" as used herein as applied to a nucleic acid, polypeptide, cell or organism refers to a nucleic acid, polypeptide, cell or organism found in nature.

As used herein, "mutation" refers to an insertion, deletion, substitution, duplication or inversion of one or more amino acids or nucleotides compared to a wild-type or reference amino acid sequence or a wild-type or reference nucleotide sequence.

As used herein, the term "isolated" is intended to describe a polynucleotide, polypeptide, or cell that is in an environment different from that in which the polynucleotide, polypeptide, or cell naturally exists. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

As used herein, "host cell" means a eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell strain) cultured as a single-cell entity that serves as a recipient of a nucleic acid (e.g., an AAV vector), and includes progeny of the original cell that has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily have exactly the same morphology or genome or total DNA complement sequence as the original parent cell due to natural, accidental, or deliberate mutations. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which a heterologous nucleic acid (e.g., an AAV vector) has been introduced.

The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in proteins with similar side chains. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; the group of amino acids with amide side chains consists of asparagine and glutamine; the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains consists of lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

As used herein, "treatment" or "treating" are used interchangeably herein and refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit and/or preventive benefit. A therapeutic benefit means eradication or amelioration of the underlying condition or disease being treated. A therapeutic benefit may also be achieved by eradication or amelioration of one or more symptoms or improvement of one or more clinical parameters associated with the underlying disease, such that improvement is observed in the individual, although the individual may still suffer from the underlying disease.

As used herein, the terms "therapeutically effective amount" and "therapeutically effective dose" refer to an amount of a drug or biological agent alone or as part of a composition that, when administered in one or repeated doses to a subject (such as a human or experimental animal), has any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristic of a disease state or condition. The effect need not be absolutely beneficial.

As used herein, "administering" means a method of giving a dose of a compound (eg, a composition of the disclosure) or a composition (eg, a pharmaceutical composition) to a subject.

“Individual” means a mammal. Mammals include, but are not limited to, domesticated animals, non-human primates, humans, dogs, rabbits, mice, rats, and other rodents.

The term "low-density lipoprotein (LDL)" refers to one of the five major types of lipoproteins, ranging from the least dense (smaller in weight to volume) to the most dense (larger in weight to volume): chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high-density lipoprotein (HDL). Lipoproteins transport lipids (fats) around the body in the extracellular fluid, thereby facilitating the transfer of fats into cell bodies via receptor-mediated endocytosis. The diameter of LDL particles is approximately 220 to 275 angstroms.

"Low-density lipoprotein (LDL) receptor" refers to the 839-amino acid receptor protein (after removal of the 21-amino acid signal peptide) that mediates the endocytosis of cholesterol-rich LDL particles. It is a cell surface receptor that recognizes apoB100 and apoE proteins found in chylomicron remnants and VLDL remnants (IDL), resulting in the binding and endocytosis of LDL-cholesterol. This process occurs in all nucleated cells, but primarily in the liver, which removes approximately 70% of LDL from circulation. The human LDLR gene is partially described in the NCBI database (ncbi.nlm.nih.gov) as reference sequence NG_009060.1, which is incorporated herein by reference.

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. I. General Methods

Unless otherwise specified, the practice of the present invention employs techniques familiar from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA as found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition (Ausubel et al., ed., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al., ed., Academic Press 1999); Viral Vectors (Kaplift and Loewy, ed., Academic Press 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle and Griffiths, John Wiley & Sons 1998), the disclosure of which is incorporated herein by reference.

Where a range of values is provided, it is understood that the endpoints are included and that each intervening value between the upper and lower limits of that range is encompassed (unless the context clearly indicates otherwise, to one-tenth of the unit of the lower limit) and any other stated or intervening value within that stated range. The upper and lower limits of such smaller ranges may independently be included in the smaller ranges and also encompassed within the stated ranges, subject to any specifically excluded limit. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure belongs. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials related to the cited publications.

It should be understood that certain features of the disclosure that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. In other cases, various features of the disclosure that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. It is intended that all combinations of embodiments of the disclosure are specifically covered by the disclosure and disclosed herein, as if each combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically covered by the disclosure and disclosed herein, as if each such subcombination were individually and explicitly disclosed herein. II. System for epigenetic modification and inhibition of the PCSK9 gene

In a first aspect, the disclosure provides a system comprising or encoding a long-term repressor fusion protein ("LTRP") comprising a DNA binding protein and a repressor domain capable of binding to a target nucleic acid sequence of a PCSK9 gene targeted for transcriptional repression, silencing and/or epigenetic modification. The disclosure also provides a system comprising or encoding a LTRP. In some cases, the system is designed to inhibit transcription of a PCSK9 gene with a mutation in a eukaryotic cell.

As used herein, "system" can be used interchangeably with "composition". The present disclosure also provides nucleic acids encoding the systems provided herein. Methods of making such systems, and methods of using such systems, including methods of gene suppression and/or epigenetic modification and methods of treating PCSK9-related diseases are also provided herein. For clarity, the term "system" also includes any encoding DNA, RNA or vector and the like that can be used to produce the suppressor fusion protein and gRNA components of the system.

In some embodiments, the DNA binding protein used in the long-term suppressor fusion protein of the present invention comprises a zinc finger (ZF) or TALE (transcription activator-like effector) protein or its DNA binding domain, also referred to herein as a DNA binding protein, which binds to the target nucleic acid but does not cleave the target nucleic acid. The DNA binding domain of TALE comprises a tandem array of customizable monomers 33-34 amino acids (aa) long, which can theoretically be assembled according to a repeat sequence combined with a base pair recognition code to recognize any gene sequence (Jain, S. et al., TALE outperforms Cas9 in editing heterochromatin target sites. Nat. Commun. 12:606 (2021)). The specificity of TALE binding to DNA is caused by two polymorphic amino acids, the so-called repeat variable diresidue (RVD), located at positions 12 and 13 of the repeat unit. By rearranging the repeat sequence, the DNA binding specificity of TALE can be changed arbitrarily. Zinc finger proteins are transcription factors, in which each zinc finger recognizes 3 to 4 bases of DNA. By mixing and matching these zinc finger modules, ZFs can be customized to the sequence to be targeted. Exemplary ZFs capable of binding to the PCSK9 gene are described in WO2018049009A2.

In some embodiments, the DNA binding protein used in the long-term suppressor fusion protein is a catalytically ineffective Class 1 or Class 2 CRISPR protein. Catalytically ineffective CRISPR proteins are also referred to in the art as "catalytically inactive" CRISPR proteins. In one embodiment, the Class 2 Type II proteins are catalytically ineffective Cas9. In another embodiment, the Class 2 Type V CRISPR proteins are selected from the group consisting of Type II, Type V, or Type VI proteins. In one embodiment, the Class 2 Type V proteins are selected from the group consisting of: Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas14, and/or CasΦ, in each case rendered catalytically ineffective by specific mutations as described herein. The CRISPR-based system further comprises a guide nucleic acid, such as a guide RNA (gRNA), having a targeting sequence complementary to a target sequence of the PCSK9 gene that is bound by and inhibited by the complex of the fusion protein (CRISPR protein and linked inhibitory domain) and the gRNA.

In some embodiments, the disclosure provides systems comprising or encoding long-term suppressor fusion proteins and guide RNA (gRNA), and nucleic acids encoding long-term suppressor fusion proteins and/or gRNA, the fusion protein comprising a catalytically inactive CasX CRISPR nuclease protein and a linked suppressor domain, the guide RNA comprising a targeting sequence complementary to a target nucleic acid sequence of a PCSK9 gene targeted for transcriptional inhibition, silencing or downregulation. In some embodiments, the system comprises a long-term suppressor fusion protein and gRNA in the form of a gene suppressor pair ("LTRP:gRNA system") disclosed herein, which is capable of forming a ribonucleoprotein (RNP) complex and binding a PCSK9 target nucleic acid. In other cases, the disclosure provides systems of nucleic acids encoding long-term suppressor fusion proteins and gRNA. In still other cases, the present disclosure provides systems of gRNA and mRNA encoding long-term suppressor fusion proteins for use in certain particle formulations (e.g., LNPs) described herein.

Also provided herein are methods of making long-term suppressor fusion proteins and gRNAs, and methods of using the LTRP:gRNA system, including methods of gene inhibition and/or epigenetic modification of the PCSK9 gene and methods of treating PCSK9- related diseases or disorders. The LTRP:gRNA system and its characteristic DNA binding proteins (e.g., dCasX) and linked suppressor domains and gRNA components, as well as delivery modes and methods of using the systems for inhibition, downregulation or silencing of the PCSK9 gene are described more fully below.

In some embodiments, the disclosure provides a system specifically designed to inhibit or silence the transcription of the PCSK9 gene. In some cases, the system is designed to inhibit the transcription of the PCSK9 gene with a gain-of-function mutation in a eukaryotic cell. In some cases, the system is designed to inhibit the transcription of the wild-type PCSK9 gene in a eukaryotic cell. In an alternative, the system is designed to inhibit the transcription of the mutant allele of the PCSK9 gene in a eukaryotic cell. In general, any portion of the PCSK9 gene can be targeted using the programmable system and method provided herein, which is described more fully herein.

The PCSK9 gene encodes proprotein convertase subtilisin/kexin type 9 ("PCSK9"), a protein that binds to a receptor on low-density lipoprotein particles (LDL) to transport LDL into cells. The PCSK9 gene covers a sequence spanning chr1:55,039,476-55,064,853 across the human genome (GRCh38/hg38) (the symbol refers to chromosome 1 (chr1)), starting from 55,039,476 bp to 55,064,853 bp on chromosome 1 (Homo sapiens Updated Annotation Release 109.20190905, GRCh38.p13) (NCBI). The human PCSK9 gene is described in part in the NCBI database (ncbi.nlm.nih.gov) as reference sequence NG_009061.1, which is incorporated herein by reference. The PCSK9 locus has 12 exons that produce a 3636 bp mRNA encoding a 692-amino acid protein that undergoes an autocatalytic cleavage reaction after its synthesis that cleaves the prodomain, thereby producing an activated protein with 540 amino acids. The prodomain remains connected to the catalytic and resistin-like domains, which may be because the prodomain acts as a chaperone and promotes folding and secretion (Seidah, NG et al., Proc Natl Acad Sci USA 100(3):928 (2003)). The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation (Seidah NG et al.). This protein, also known as neural apoptosis-regulated convertase, is a serine protease belonging to the proteinase K subfamily of Bacillus subtilisin.

The human PCSK9 gene (HGNC:20001) encodes a protein (Q8NBP7) with the following sequence: III. Proteins that inhibit catalytic inactivation of subsystems

In some embodiments, the DNA binding protein used in the long-term suppressor fusion proteins of the present disclosure is a zinc finger (ZF) or TALE (transcription activator-like effector) protein that can bind but not cleave PCSK9 target nucleic acid.

In some embodiments, the DNA binding protein is a catalytically ineffective Class 1 or Class 2 CRISPR protein. In some embodiments, the DNA binding protein is a Class 2 Type II CRISPR protein. In one embodiment, the Class 2 Type II protein is a catalytically ineffective Cas9. In another embodiment, the Class 2 CRISPR protein is selected from the group consisting of Type II, Type V, or Type VI proteins. In one embodiment, the Class 2 CRISPR Type V protein is selected from the group consisting of: Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas14, and/or CasΦ, in each case rendered catalytically ineffective by specific mutations as described herein. In another embodiment, the Class 2 type V CRISPR protein is a catalytically inactive CasX protein.

The CasX proteins (including dCasX) disclosed herein comprise the following domains: non-target strand binding (NTSB) domain, target strand loading (TSL) domain, helix I domain, helix II domain, oligonucleotide binding domain (OBD) and RuvC domain, and in some cases, the domains can be further divided into subdomains, as listed in Table 1.

In the context of the present disclosure, the CasX used in these systems is catalytically inactive (dCasX); this is achieved by introducing mutations at selected positions in the RuvC sequence, as described below. a. Reference CasX protein

The present disclosure provides naturally occurring CasX proteins (referred to herein as "reference CasX proteins") that are subsequently modified to generate the engineered dCasX of the present disclosure. For example, the reference CasX protein can be isolated from naturally occurring prokaryotes, such as Deltaproteobacteria , Planctomycetes , or Candidatus Sungbacteria species . The reference CasX protein (interchangeably referred to herein as a reference CasX polypeptide) is a Class 2 V-type CRISPR/Cas endonuclease that belongs to the CasX (interchangeably referred to as Cas12e) family of proteins that interact with a guide RNA to form a ribonucleoprotein (RNP) complex.

In some cases, the reference CasX protein is isolated or derived from Deltaproteobacteria and comprises the sequence of SEQ ID NO: 1.

In some cases, the reference CasX protein is isolated or derived from Planctomycetes and comprises the sequence of SEQ ID NO: 2.

In some cases, the reference CasX protein is isolated or derived from C. sonnei sp. and comprises the sequence of SEQ ID NO: 3. b. Catalytically inactive Class 1 or Class 2 CRISPR proteins

In long-term suppressor fusion proteins and gene suppressor systems comprising the long-term suppressor fusion proteins of the present disclosure, the catalytically ineffective Class 1 or Class 2 CRISPR protein is catalytically ineffective in that it cannot cleave DNA, but retains the ability to bind target nucleic acids when complexed with a guide RNA (gRNA). The present disclosure provides catalytically ineffective variants of Class 1 or Class 2 CRISPR proteins, wherein the catalytically ineffective variants comprise multiple modifications in a selected domain. The present disclosure provides catalytically ineffective CasX variants (interchangeably referred to herein as "dCasX variants" or "dCasX variant proteins"), wherein the catalytically ineffective CasX variants comprise multiple modifications in the RuvC domain relative to a catalytically ineffective version of a reference CasX protein comprising the sequence of SEQ ID NOs: 1 to 3 (see above). In some embodiments, the catalytically ineffective reference CasX protein comprises a substitution at residue 672, 769 and/or 935 relative to SEQ ID NO: 1. In some embodiments, the catalytically ineffective reference CasX protein comprises a substitution at residue 672, 769 and/or 935 relative to SEQ ID NO: 1. In some embodiments, the catalytically ineffective reference CasX protein comprises a substitution at residue 672, 769 and/or 935 relative to SEQ ID NO: 1. In other embodiments, the catalytically ineffective reference CasX protein comprises a substitution at amino acid 659, 756 and/or 922 relative to SEQ ID NO: 2. In some embodiments, the catalytically ineffective reference CasX protein comprises a substitution at residue 659, 756 and/or 922 relative to SEQ ID NO: 2. Exemplary RuvC domains of dCasX disclosed herein include amino acids 661-824 and 935-986 of SEQ ID NO: 1, or amino acids 648-812 and 922-978 of SEQ ID NO: 2, having one or more amino acid modifications relative to the RuvC cleavage domain sequence, wherein the dCasX variant exhibits one or more improved characteristics compared to the reference dCasX. In other embodiments, the catalytically ineffective CasX variant protein comprises a deletion of all or part of the RuvC domain of the reference CasX protein. It should be understood that the same aforementioned substitutions or deletions can be similarly introduced into CasX variants known in the art to generate dCasX variants (for exemplary sequences, see, e.g., WO2022120095A1 and US11,560,555, which are incorporated herein by reference).

In some embodiments, a long-term suppressor fusion protein comprising a dCasX variant and a linked suppressor domain exhibits at least one improved feature compared to a long-term suppressor fusion protein comprising a reference dCasX protein and a similar linked suppressor domain. All dCasX variants that improve one or more functions or features of a long-term suppressor fusion protein comprising a dCasX variant protein and a linked suppressor domain compared to a long-term suppressor fusion protein comprising a reference dCasX protein are contemplated to be within the scope of the present disclosure. In some embodiments, the modification is a mutation of one or more amino acids of the reference dCasX (other than those amino acids that render the dCasX catalytically ineffective). For example, a dCasX variant may comprise one or more amino acid substitutions, insertions, deletions, or exchange domains, or any combination thereof, relative to a reference dCasX protein sequence. Any amino acid may be substituted with any other amino acid in the substitutions described herein. The substitution may be a conservative substitution (e.g., a basic amino acid is substituted with another basic amino acid). The substitution may be a non-conservative substitution (e.g., a basic amino acid is substituted with an acidic amino acid or vice versa). For example, proline in a reference dCasX protein may be substituted with any of the following to generate a dCasX variant protein of the present disclosure: arginine, histidine, lysine, aspartic acid, glutamine, serine, threonine, aspartic acid, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, or valine. In some embodiments, dCasX variants exhibit improved properties compared to a reference dCasX. Exemplary improved features of dCasX variant embodiments include, but are not limited to: improved variant folding, increased binding affinity for target nucleic acids, improved ability to utilize a wider range of PAM sequences in transcriptional repression and/or binding of target nucleic acids, improved target DNA unfolding, increased target strand loading, increased binding of non-target strands of DNA, improved protein stability, increased ability to form complexes with gRNAs, increased binding affinity for gRNAs, improved protein:gRNA (RNP) complex stability, and increased repressor activity when linked to a repressor domain and complexed to RNPs, improved repressor specificity for target nucleic acids, reduced off-target repression, and an increase in the percentage of eukaryotic genomes that can be effectively repressed and/or epigenetically modified. In some embodiments, the improved characteristics of a dCasX variant are improved by at least about 1.1 to about 100,000 fold relative to a reference dCasX protein. In some embodiments, the improved characteristics of a dCasX variant are improved by at least about 1.1 to about 10,000 fold, improved by at least about 1.1 to about 1,000 fold, improved by at least about 1.1 to about 500 fold, improved by at least about 1.1 to about 400 fold, improved by at least about 1.1 to about 300 fold, improved by at least about 1.1 to about 200 fold, improved by at least about 1.1 to about 100 fold, improved by at least about 1.1 to about 50 fold, improved by at least about 1.1 to about 40 fold, improved by at least about 1.1 to about 30 fold, improved by at least about 1.1 to about 20 fold, improved by at least about 1.1 to about 10 fold, improved by at least about 1.1 to about In some embodiments, the improved characteristics of the dCasX variants are improved by at least about 10 to about 1000 times relative to a reference dCasX protein. Additional disclosure regarding the improved features is described below.

In other embodiments, the modification is the replacement of one or more domains of the reference dCasX with one or more domains from a different CasX. In some embodiments, the insertion comprises the insertion of part or all of a domain from a different CasX protein. Mutations may be placed in any one or more domains of the dCasX variant, and may include, for example, a deletion of part or all of one or more domains, or one or more amino acid substitutions, deletions, or insertions in any domain. The domains of the dCasX protein include a non-target strand binding (NTSB) domain, a target strand loading (TSL) domain, a helical I domain, a helical II domain, an oligonucleotide binding domain (OBD), and a RuvC DNA cleavage domain, which may further include subdomains described below.

In some embodiments, the dCasX variant protein comprises 800 to 1100 amino acids or 900 to 1000 amino acids.

Compared to RNPs comprising a fusion protein of a reference dCasX protein and a gRNA in a comparable assay system, the long-term suppressor fusion proteins comprising a dCasX variant and a linked suppressor domain disclosed herein have enhanced ability to effectively bind to a target nucleic acid when complexed with a gRNA as an RNP using and bound to a PAM TC motif (including a PAM sequence selected from TTC, ATC, GTC, or CTC). In the foregoing, the PAM sequence is located at least 1 nucleotide 5' from the non-target strand of the protospacer having the same identity as the targeting sequence of the gRNA.

In some embodiments, the disclosed RNPs comprising a long-term suppressor fusion protein comprising a dCasX variant and a linked inhibitory domain and a gRNA are capable of binding a double-stranded DNA target with an efficiency of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% at a concentration of 20 pM or less. In one embodiment, the RNPs comprising a dCasX variant and a long-term suppressor fusion protein linked to an inhibitory domain and a gRNA variant exhibit greater binding to a target sequence in a target nucleic acid, wherein the PAM sequence of the target nucleic acid is TTC, compared to RNPs comprising a comparable long-term suppressor fusion protein and a gRNA containing a reference dCasX protein and a linked inhibitory domain in a comparable assay system. In another embodiment, the RNPs comprising a dCasX variant and a long-term suppressor fusion protein and a gRNA variant linked to a suppressor domain exhibit greater binding affinity to a target sequence in a target nucleic acid, wherein the PAM sequence of the target nucleic acid is ATC, compared to RNPs comprising a comparable long-term suppressor fusion protein and a gRNA containing a reference dCasX protein and a linked suppressor domain in a comparable assay system, wherein the PAM sequence of the target nucleic acid is ATC. In another embodiment, the RNPs comprising a dCasX variant and a long-term suppressor fusion protein and a gRNA variant linked to a suppressor domain exhibit greater binding affinity to a target sequence in a target nucleic acid, wherein the PAM sequence of the target nucleic acid is CTC, compared to RNPs comprising a comparable long-term suppressor fusion protein and a gRNA containing a reference dCasX protein and a linked suppressor domain in a comparable assay system. In another embodiment, the RNPs comprising a dCasX variant and a long-term suppressor fusion protein and a gRNA variant linked to a suppressor domain exhibit greater binding affinity to a target sequence in a target nucleic acid, wherein the PAM sequence of the target nucleic acid is GTC, compared to RNPs comprising a reference dCasX protein and a comparable long-term suppressor fusion protein and a gRNA linked to a suppressor domain in a comparable assay system. In other embodiments, the RNPs comprising a dCasX variant and a suppressor fusion protein and a gRNA linked to a suppressor domain exhibit greater binding affinity to a target sequence in a target nucleic acid, wherein the PAM sequence of the target nucleic acid is GTC, TTC, ATC, or CTC, compared to RNPs comprising a reference dCasX protein and a comparable suppressor fusion protein and a gRNA linked to a suppressor domain in a comparable assay system. In the aforementioned embodiments, the increased binding affinity for one or more PAM sequences is at least 1.5 times greater than the binding affinity of any of the reference dCasX proteins (modified from SEQ ID NOs: 1-3) and the gRNAs of SEQ ID NOs: 1731-1743 of Table 8 to the PAM sequences. c. dCasX variant proteins with domains from multiple source proteins

In certain embodiments, the present disclosure provides a chimeric dCasX variant protein for use in a long-term suppressor fusion protein.

As used herein, a "chimeric dCasX" protein refers to a catalytically ineffective CasX protein containing at least two domains from different sources and a catalytically ineffective CasX protein containing at least one domain that is itself in a chimeric form. Thus, in some embodiments, a chimeric dCasX protein is a chimeric dCasX protein comprising at least two domains separated or derived from different sources, such as domains from two different naturally occurring CasX proteins (e.g., from two different CasX reference proteins). In other embodiments, a chimeric dCasX protein is a protein containing at least one domain in the form of a chimeric domain, for example, in some embodiments, a portion of a domain comprises a substitution from a different CasX protein (from a reference CasX protein or another dCasX protein).

In some embodiments, at least one chimeric domain may be any of the NTSB, TSL, helix I, helix II, OBD, or RuvC domains described herein. In the case of split or non-contiguous domains such as helix I, RuvC, and OBD, a portion of the non-contiguous domain may be replaced with a corresponding portion from any other source. In some embodiments, the helix I-II domain (sometimes referred to as helix I-a) of dCasX derived from SEQ ID NO: 2 is replaced with the corresponding helix I-II sequence from SEQ ID NO: 1 to obtain a chimeric dCasX protein.

In some embodiments, the helix I-II domain and the NTSB domain of dCasX derived from SEQ ID NO: 2 are replaced by the corresponding helix I-II and NTSB sequences from SEQ ID NO: 1 to obtain a chimeric dCasX protein.

The chimeric dCasX variant protein may comprise the NTSB, TSL, helix II, helix I-II, helix II, OBD-I, and OBD-II domains from the CasX protein of SEQ ID NO: 2, and the RuvC-I and/or RuvC-II domains from the CasX protein of SEQ ID NO: 1, or vice versa, wherein mutations or other sequence changes are introduced to generate a catalytically ineffective variant with improved properties of the variant relative to the reference dCasX protein. As an example of the foregoing, the chimeric RuvC domain comprises amino acids 660 to 823 of SEQ ID NO: 1 and amino acids 921 to 978 of SEQ ID NO: 2. As an alternative example of the foregoing, the chimeric RuvC domain comprises amino acids 647 to 810 of SEQ ID NO: 2 and amino acids 934 to 986 of SEQ ID NO: 1. In a specific embodiment, the dCasX used in the long-term suppressor fusion protein comprises the NTSB domain and the Helical I-II domain from SEQ ID NO: 1 and the Helical II domain from SEQ ID NO: 2; the latter being a chimeric domain, it being understood that the dCasX variants have additional amino acid changes at selected positions (relative to the reference sequence) and that the resulting chimeric dCasX protein has improved characteristics relative to the reference dCasX protein. Sequences in Table 2 having an NTSB domain and a helical I-II domain from SEQ ID NO: 1 and a helical II domain from SEQ ID NO: 2 include dCasX 491 (SEQ ID NO: 4), 515 (SEQ ID NO: 6), 516 (SEQ ID NO: 7), 518-520 (SEQ ID NO: 9-11), 522-527 (SEQ ID NO: 12-17), 532 (SEQ ID NO: 22), 593 (SEQ ID NO: 25), 676 (SEQ ID NO: 28, with a L169K substitution in the NTSB domain), and 812 (SEQ ID NO: 29). The coordinates of the CasX domains in the reference CasX proteins of SEQ ID NO: 1 and SEQ ID NO: 2 are provided in Table 1 below. Those skilled in the art will appreciate that the domain boundaries indicated in Table 1 below are approximate, and that protein fragments whose boundaries differ from those given in the table below by 1, 2, or 3 amino acids may have the same activity as the domains described below. Table 1 : Domain coordinates in reference CasX proteins Domain Name Coordinates in SEQ ID NO: 1 * Coordinates in SEQ ID NO: 2 * OBD-I 1-55 1-57 Helix II 56-99 58-101 NTSB 100-190 102-191 Helix I-II 191-331 192-332 Helix II 332-508 333-500 OBD-II 509-659 501-646 RuvC-I 660-823 647-810 TSL 824-933 811-920 RuvC-II 934-986 921-978 *Amino acid position

In some embodiments, the dCasX variant protein used in the long-term suppressor fusion protein of the present disclosure comprises a sequence selected from the group consisting of SEQ ID NOs: 4 to 29 as described in Table 2, wherein the sequence comprises a RuvC domain comprising one or more mutations that inactivate the cleavage activity of the RuvC domain. In other embodiments, the dCasX variant protein used in the long-term suppressor fusion protein of the present disclosure comprises a sequence that is at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or at least about 99.5% identical to the sequences of SEQ ID NOs: 4 to 29 set forth in Table 2. In some embodiments, the dCasX variant protein comprises a RuvC domain comprising one or more mutations that inactivate the cleavage activity of the RuvC domain. In some embodiments, the long-term suppressor fusion protein comprising dCasX retains the ability to form RNPs with gRNA. In a specific embodiment, the dCasX variant protein in the long-term suppressor fusion protein used in the gene suppressor system of the present disclosure comprises the sequence of SEQ ID NO: 4 (dCasX 491). In another specific embodiment, the dCasX variant protein in the long-term suppressor fusion protein used in the gene suppressor system of the present disclosure comprises the sequence of SEQ ID NO: 6 (dCasX 515). In another specific embodiment, the dCasX variant protein in the long-term suppressor fusion protein used in the gene suppressor system of the present disclosure comprises the sequence of SEQ ID NO: 29 (dCasX 812). Table 2 : dCasX variant sequences SEQ ID NO dX Amino acid sequence 4 dCasX491 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 5 dCasX514 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKL ANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTIHTSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 6 dCasX515 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKL ANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 7 dCasX516 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNHNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 8 dCasX517 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGAPVGKALSDACMGTIASFLSK YQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVF WQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 9 dCasX518 RQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGN LTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSK YQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVF WQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 10 dCasX519 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKL ANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHIQLRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 11 dCasX520 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTTQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 12 dCasX522 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKRSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 13 dCasX523 QEIKRINKIRRRLVKDSNTKKAGKTYPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 14 dCasX524 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTIHSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 15 dCasX525 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAATQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 16 dCasX526 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 17 dCasX527 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGN LTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSK YQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVF WQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 18 dCasX528 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASYPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKL ANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 19 dCasX529 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASNPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKL ANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 20 dCasX530 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGWGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV twenty one dCasX531 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGYGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV twenty two dCasX532 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGN LTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSK YQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVF WQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV twenty three dCasX533 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGN LTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASYPVGKALSDACMGTIASFLSK YQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVF WQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV twenty four dCasX535 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASSPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKL ANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 25 dCasX593 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRWWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKL ANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 26 dCasX668 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGN LTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASSPVGKALSDACMGTIASFLSK YQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVF WQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 27 dCasX672 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLIKLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASSPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKL ANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 28 dCasX676 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGN LTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLIKLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASSPVGKALSDACMGTIASFLSK YQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVF WQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 29 dCasX812 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKKFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKL ANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADD MVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV d. Affinity for gRNA

In some embodiments, the long-term suppressor fusion protein comprising dCasX and a linked suppressor domain has improved affinity for gRNA relative to a comparable long-term suppressor fusion protein comprising a reference dCasX protein and a corresponding linked suppressor domain, thereby forming a ribonucleoprotein complex (RNP). The increased affinity of the long-term suppressor fusion protein for gRNA can, for example, result in a lower _Kd for the production of the RNP complex, which in some cases can lead to more stable RNP complex formation. In some embodiments, the long-term suppressor fusion protein has a _Kd for the gRNA that is increased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold relative to a reference dCasX protein and a linked suppressor domain. In some embodiments, the long-term suppressor fusion protein comprising the dCasX variant has an increased binding affinity for the gRNA by about 1.1 to about 10 fold compared to the corresponding suppressor fusion protein comprising a catalytically ineffective variant of a reference CasX protein of SEQ ID NO: 2.

In some embodiments, the increased affinity of the long-term suppressor fusion protein for the gRNA increases the stability of the ribonucleoprotein complex when delivered to mammalian cells (including delivery to an individual in vivo). When delivered to an individual, this increased stability can affect the function and utility of the complex in the individual's cells, as well as improve the pharmacokinetic properties in the blood. In some embodiments, the increased affinity of the long-term suppressor fusion protein and the resulting increased stability of the ribonucleoprotein complex allow lower doses of the long-term suppressor fusion protein to be delivered to an individual or cell while still having the desired activity; for example, in vivo or in vitro gene suppression and/or epigenetic modification. The improved ability to form RNPs and keep them in a stable form can be assessed using in vitro assays known in the art.

In some embodiments, when both the long-term suppressor fusion protein and the gRNA are retained in the RNP complex, the higher affinity (tighter binding) of the dCasX variant protein of the long-term suppressor fusion protein to the gRNA allows for a greater amount of transcriptional inhibition and/or epigenetic modification events. The increase in transcriptional inhibition events can be assessed using the assays described herein.

Methods for measuring the binding affinity of a long-term suppressor fusion protein for a gRNA include in vitro methods using purified long-term suppressor fusion proteins and gRNAs. If the gRNA or long-term suppressor fusion protein is fluorophore-labeled, the binding affinity of the long-term suppressor fusion protein can be measured by fluorescence polarization. Alternatively or additionally, binding affinity can be measured by biointerferometry, electrophoretic mobility shift assay (EMSA), or filter binding. Additional standard techniques for quantifying the absolute affinity of an RNA binding protein (such as the dCasX protein disclosed herein) for a specific gRNA (such as a reference gRNA and its variants) include, but are not limited to, isothermal calorimetry (ITC) and surface plasmon resonance (SPR). e. Improved specificity for target nucleic acid sequences

In some embodiments, a long-term suppressor fusion protein comprising a dCasX variant protein and an associated suppressor domain has improved specificity for a target nucleic acid sequence complementary to the targeting sequence of the gRNA relative to a reference dCasX protein associated with a suppressor domain. As used herein, "specificity," sometimes referred to as "target specificity," refers to the degree to which a CRISPR/Cas system ribonucleoprotein complex binds to off-target sequences that are similar but not identical to the target nucleic acid sequence; for example, a long-term suppressor fusion protein containing an RNP with a high degree of specificity will exhibit reduced sequence off-target methylation relative to an RNP of a reference dCasX associated with a suppressor domain. The specificity of a long-term suppressor fusion protein and the reduction of potential deleterious off-target effects can help achieve an acceptable therapeutic index for use in mammalian subjects. Without wishing to be bound by theory, amino acid changes in the helical I and II domains can increase the specificity of the long-term suppressor fusion protein for the target nucleic acid strand, and can thereby increase the specificity of the long-term suppressor fusion protein for the target nucleic acid overall. In some embodiments, amino acid changes that increase the specificity of the long-term suppressor fusion protein for the target nucleic acid can also reduce the affinity of the long-term suppressor fusion protein for DNA, but the overall benefit and safety of the composition are improved. f. Suppressor Fusion Proteins with Heterologous Proteins

Also contemplated within the scope of the present disclosure are long-term suppressor fusion proteins comprising a heterologous protein fused to a long-term suppressor fusion protein for use in the systems of the present disclosure. This includes long-term suppressor fusion proteins comprising fusion to the N-terminus or C-terminus of a heterologous protein or domain thereof. In some embodiments, a long-term suppressor fusion protein is fused to one or more proteins or domains thereof having different activities of interest.

In some cases, the heterologous polypeptide (fusion partner) for use with the long-term suppressor fusion protein provides secondary cellular localization, i.e., the heterologous polypeptide contains a secondary cellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus; a sequence that keeps the fusion protein outside the nucleus, such as a nuclear export sequence (NES); a sequence that keeps the fusion protein in the cytoplasm; a mitochondrial localization signal for targeting to mitochondria; a chloroplast localization signal for targeting to chloroplasts; an ER retention signal; and the like).

In some cases, the long-term inhibitor fusion protein includes a nuclear localization signal (NLS) (fused to the NLS). In some cases, the long-term inhibitor fusion protein is fused to 2 or more, 3 or more, 4 or more, or 5 or more, 6 or more, 7 or more, 8 or more NLS. In some cases, one or more NLS (2 or more, 3 or more, 4 or more, or 5 or more NLS) is located at or near the N-terminus and/or C-terminus of the long-term inhibitor fusion protein (e.g., within a range of 20 amino acids). In some cases, one or more NLS (2 or more, 3 or more, 4 or more, or 5 or more NLS) is located at or near the N-terminus of the long-term inhibitor fusion protein (e.g., within a range of 20 amino acids). In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are located at or near (e.g., within 20 amino acids) the C-terminus of a long-term suppressor fusion protein. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are located at or near (e.g., within 20 amino acids) the N-terminus and/or C-terminus of a long-term suppressor fusion protein. In some cases, a single NLS is located at the N-terminus of a long-term suppressor fusion protein and a single NLS is located at the C-terminus. It will be appreciated by those skilled in the art that an NLS located at or near the N-terminus or C-terminus of a protein may be within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids from the N-terminus or C-terminus. In some embodiments, the NLS attached to the N-terminus of a dCasX or long-term suppressor fusion protein is the same as the NLS attached to the C-terminus. In other embodiments, the NLS attached to the N-terminus of a dCasX or long-term suppressor fusion protein is different from the NLS attached to the C-terminus. Representative configurations of long-term suppressor fusion proteins with NLSs are shown in FIG. 61 . In some embodiments, NLSs suitable for use with long-term suppressor fusion proteins in the systems of the present disclosure include sequences that are at least about 85%, at least about 90%, or at least about 95% identical to or identical to sequences derived from: the NLS of the large T antigen of the SV40 virus, having the amino acid sequence PKKKRKV (SEQ ID NO: 30); an NLS from a nucleoplasmic protein (e.g., a nucleoplasmic protein bipartite NLS, having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 31)); a c-MYC NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 32) or RQRRNELKRSP (SEQ ID NO: 33). In some embodiments, the NLS and short peptide linker attached to the N-terminus of the long-term suppressor fusion protein are the sequence PKKKRKVSR (SEQ ID NO: 34). In some embodiments, the NLS and short peptide linker attached to the N-terminus of the long-term suppressor fusion protein is the sequence PKKKRKVSRVNGSGSGGG (SEQ ID NO: 3298). In some embodiments, the NLS and short peptide linker attached to the C-terminus of the long-term suppressor fusion protein is the sequence TSPKKKRKV (SEQ ID NO: 3273). In some embodiments, the NLS attached to the N-terminus of the long-term suppressor fusion protein is selected from the group consisting of the N-terminal sequences described in Table 3. In some embodiments, the NLS comprises a sequence selected from the group consisting of SEQ ID NOs: 30 to 97, 3273 and 3298. In some embodiments, the NLS comprises a sequence selected from the group consisting of SEQ ID NOs: 34 to 97. In some embodiments, the NLS linked to the C-terminus of the long-term suppressor fusion protein is selected from the group consisting of the C-terminal sequences described in Table 4. In some embodiments, NLSs suitable for use with long-term suppressor fusion proteins in the systems of the present disclosure include sequences that are at least about 80%, at least about 90%, or at least about 95% identical or identical to one or more of the sequences in Table 3 or Table 4. It should be understood by those of ordinary skill in the art that any of the NLS sequences described in Tables 3 and 4 can be fused to or near the N-terminus or C-terminus of the long-term suppressor fusion proteins described herein. Table 3 : N- terminal NLS amino acid sequences NLS amino acid sequence * SEQ ID NO PKKKRKV SR 34 PKKKRKV GGS PKKKRKV GGS PKKKRKV GGS PKKKRKV SR 35 PKKKRKV GGS PKKKRKV GGS PKKKRKV GGS PKKKRKV GGS PKKKRKV GGS PKKKRKV SR 36 PAAKRVKLD SR 37 PAAKRVKLD GGS PAAKRVKLD SR 38 PAAKRVKLD GGS PAAKRVKLD GGS PAAKRVKLD GGS PAAKRVKLD SR 39 PAAKRVKLD GGS PAAKRVKLD GGS PAAKRVKLD GGS PAAKRVKLD GGS PAAKRVKLD GGS PAAKRVKLD SR 40 KRPAATKKAGQAKKKK SR 41 KRPAATKKAGQAKKKK GGS KRPAATKKAGQAKKKK SR 42 PAAKRVKLD GGS PKKKRKV SR 43 PAAKKKKLD GGS PKKKRKV SR 44 PAAKKKKLD SR 45 PAAKKKKLD GGS PAAKKKKLD GGS PAAKKKKLD SR 46 PAAKKKKLD GGS PAAKKKKLD GGS PAAKKKKLD GGS PAAKKKKLD SR 47 PAKRARRGYKC SR 48 PAKRARRGYKC GS PAKRARRGYKC SR 49 PRRKREE SR 50 PYRGRKE SR 51 PLRKRPRR SR 52 PLRKRPRR GS PLRKRPRR SR 53 PAAKRVKLD GG KRTADGSEFESPKKKRKV GGS 54 PAAKRVKLD GG KRTADGSEFESPKKKRKV PPPPG 55 PAAKRVKLD GG KRTADGSEFESPKKKRKV GIHGVPAAPG 56 PAAKRVKLD GG KRTADGSEFESPKKKRKV GGGSGGGSPG 57 PAAKRVKLD GG KRTADGSEFESPKKKRKV PGGGGSGGGSPG 58 PAAKRVKLD GG KRTADGSEFESPKKKRKV AEAAAKEAAAKEAAAKAPG 59 PAAKRVKLD GGS PKKKRKV GGS 60 PAAKRVKLD PPP PKKKRKV PG 61 PAAKRVKLD PG 62 PAAKRVKLD GGGSGGGSGGGS 63 PAAKRVKLD PPP 64 PAAKRVKLD GGGSGGGSGGGSPPP 65 PKKKRKV PPP 66 PKKKRKV GGS 67 *The residues in bold are NLS residues, while the residues in non-bold are linkers. Table 4 : C- terminal NLS amino acid sequences NLS amino acid sequence SEQ ID NO GS PKKKRKV GGS PKKKRKV GGS PKKKRKV GGS PKKKRKV 68 GS PKKKRKV GGS PKKKRKV GGS PKKKRKV GGS PKKKRKV GGS PKKKRKV GGS PKKKRKV 69 GS PAAKRVKLD GGS PAAKRVKLD 70 GS PAAKRVKLD GGS PAAKRVKLD GGS PAAKRVKLD GGS PAAKRVKLD 71 GS KRPAATKKAGQAKKKK 72 KRPAATKKAGQAKKKK GGS KRPAATKKAGQAKKKK 73 GS KLGPRKATGRW GS 74 GS KRKGSPERGERKRHW GS 75 GS PKKKRKV GSGS KRPAATKKAGQAKKKK LE 76 GP KRTADSQHSTPPKTKRKV EFE PKKKRKV 77 GGGSGGGSKRTADSQHSTPPKTKRKV EFE PKKKRKV 78 AEAAAKEAAAKEAAAKA KRTADSQHSTPPKTKRKV EFE PKKKRKV 79 GPPKKKRKVGGS KRTADSQHSTPPKTKRKV EFE PKKKRKV 80 GPAEAAAKEAAAKEAAAKA PAAKRVKLD 81 GPGGGSGGGSGGGS PAAKRVKLD 82 GP PAAKRVKLD 83 VGS KRPAATKKAGQAKKKK 84 TGGGPGGGAAAGSGS PKKKRKV GSGS KRPAATKKAGQAKKKK LE 85 TGGGPGGGAAAGSGS PKKKRKV GSGS 86 PPP PKKKRKV PPP 87 GGS PKKKRKV PPP 88 PPP PKKKRKV 89 GGS PKKKRKV 90 GGS PKKKRKV GGSGGSGGS 91 GGS PKKKRKV GGSPKKKRKV 92 GGSGGSGGS PKKKRKV GGS PKKKRKV 93 VGGGSGGGSGGGS PAAKRVKLD 94 VPPP PAAKRVKLD 95 VPPPGGGSGGGSGGGS PAAKRVKLD 96 VGS PAAKRVKLD 97

In some embodiments, one or more NLSs are linked to a long-term suppressor fusion protein or an adjacent NLS via an optional linker peptide. In some embodiments, the linker peptide is selected from the group consisting of SR, GS, GP, TS, VGS, GGS, (G)n (SEQ ID NO: 98), (GS)n (SEQ ID NO: 99), (GSGGS)n (SEQ ID NO: 100), (GGSGGS)n (SEQ ID NO: 101), (GGGS)n (SEQ ID NO: 102), GGSG (SEQ ID NO: 103), GGSGG (SEQ ID NO: 104), GSGSG (SEQ ID NO: 105), GSGGG (SEQ ID NO: 106), GGGSG (SEQ ID NO: 107), GSSSG (SEQ ID NO: 108), GPGP (SEQ ID NO: 109), GGP, PPP, VPPP, PPAPPA (SEQ ID NO: 110), PPPG (SEQ ID NO: 111), PPPGPPP (SEQ ID NO: 112). 112), PPP(GGGS)n (SEQ ID NO: 113), (GGGS)nPPP (SEQ ID NO: 114), AEAAAKEAAAKEAAAKA (SEQ ID NO: 115), VPPPPGGGSGGGSGGGS (SEQ ID NO: 116), TGGGPGGGAAAGSGS (SEQ ID NO: 117), GGGSGGGSGGGGSPPP (SEQ ID NO: 118), TPPKTKRKVEFE (SEQ ID NO: 119), GGSGGGS (SEQ ID NO: 120), GGSGSGG (SEQ ID NO: 121), SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 122), GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE (SEQ ID NO: 123) and GGSGGG (SEQ ID NO: 124), wherein n is 1 to 5.

Generally, the NLS (or multiple NLSs) are strong enough to drive the accumulation of the long-term suppressor fusion protein in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus can be performed by any suitable technique. For example, a detectable marker can be fused to the long-term suppressor fusion protein so that its location within the cell can be observed. The nucleus can also be isolated from the cell, and its contents can then be analyzed by any method suitable for detecting proteins, such as immunohistochemistry, Western blotting, or enzyme activity analysis. Accumulation in the nucleus can also be determined indirectly. IV. Long-term suppressor domain fusion proteins

The present disclosure provides a system comprising a long-term fusion protein of a DNA binding protein linked to multiple inhibitory subdomains, wherein the system is capable of binding to a PCSK9 target nucleic acid and inhibiting transcription of the PCSK9 gene, including inhibition by epigenetic modification of the target nucleic acid. Exemplary DNA binding proteins for fusion proteins include zinc fingers (ZF), TALE (transcription activator-like effector) proteins, and catalytically inactive CRISPR proteins.

In some embodiments, the disclosure provides a system of long-term suppressor fusion proteins that comprise a catalytically inactive CRISPR protein (such as dCasX) linked to multiple suppressor domains when complexed with a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence of PCSK9 , wherein the system is capable of binding to a target nucleic acid of PCSK9 and inhibiting or silencing transcription of the PCSK9 gene. Examples of gene suppression processes that reduce transcription include, but are not limited to, processes that inhibit the formation of a transcription initiation complex, processes that reduce the rate of transcription initiation, processes that reduce the rate of transcription elongation, processes that reduce the persistence of transcription, and processes that antagonize transcriptional activation (e.g., by blocking the binding of a transcriptional activating factor). Gene suppression can constitute, for example, prevention of activation and suppression of expression below the current amount. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription; the latter can be caused by epigenetic modifications of the target nucleic acid.

Among the repressor domains that can repress or silence genes, the Krüppel-associated box (KRAB) repressor domain is the most efficient in the human genome system (Alerasool, N., et al. An efficient KRAB domain for CRISPRi applications. Nat. Methods 17:1093 (2020)). KRAB-like domains exist in approximately 400 human zinc finger protein-based transcription factors, which can recruit additional repressor domains such as but not limited to Trim28 (also known as Kap1 or Tif1-β) when the attached dCasX binds to the target nucleic acid. These repressor domains in turn assemble protein complexes with chromatin regulatory factors such as CBX5/HP1α and SETDB1 to induce gene transcription repression, but the repression is also time-limited. Representative, non-limiting examples of KRAB domains suitable for use in the systems of the present disclosure include ZIM3 (SEQ ID NO: 129) and ZNF10 (SEQ ID NO: 128). The present disclosure provides additional repressor domains from human sources, as well as repressor domains from non-human sources with significantly different sequences (referred to herein as "RD1"), which have been found to cause enhanced transcriptional repression compared to ZIM3 and ZNF10 when incorporated into the long-term repressor fusion protein construct embodiments described herein.

In some embodiments, the present disclosure provides systems in which the modifications imparted by using the LTRP:gRNA system are epigenetic modifications, and thus, PCSK9 gene silencing can be inherited by mechanisms other than the replication of the edited DNA. As used herein, "epigenetic modification" means a modification of DNA or histones associated with DNA, rather than a change (e.g., substitution, deletion, or rearrangement) in the DNA sequence itself, wherein the modification is a direct modification by a component of the system or an indirect modification by recruiting one or more additional cellular components, but wherein the DNA target nucleic acid sequence itself is not edited to change the sequence. For example, DNA methyltransferase 3A (DNMT3A) (or its catalytic domain) directly modifies DNA through DNA methylation, while KRAB recruits the KAP-1/TIF1β co-repressor complex, which acts as a potent transcriptional repressor and can further recruit factors associated with DNA methylation and repressive chromatin formation, such as heterochromatin protein 1 (HP1), histone deacetylases, and histone methyltransferases (Ying, Y. et al., The Krüppel-associated box repressor domain induces reversible and irreversible regulation of endogenous mouse genes by mediating different chromatin states. Nucleic Acids Res. 43(3): 1549 (2015)). In addition, the catalytically inactive DNMT3L co-factor, together with the cell's endogenous DNMT1, helps to establish heritable methylation patterns after DNA replication. The ATRX-DNMT3-DNMT3L domain (ADD) of DNMT3A is known to have two key functions: 1) it allosterically regulates the catalytic activity of DNMT3A by acting as a methyltransferase autoinhibitory domain, and 2) it specifically interacts with the unmethylated histone H3 tail at lysine (K)4, resulting in preferential methylation of DNA bound to the unmethylated chromatin H3 tail at K4 (Zhang, Y. et al., Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Research 38:4246 (2010)). In some embodiments, the included ADD domain enhances transcriptional repression of a targeted gene when incorporated into a LTRP compared to an otherwise identical LTRP lacking the ADD domain. In other embodiments, the included ADD domain enhances the specificity of transcriptional inhibition of the targeted gene when compared to an otherwise identical LTRP lacking the ADD domain. Supporting data for the foregoing are provided in the Examples and in WO2023049742A2, which is incorporated herein by reference.

In some embodiments, the long-term suppressor fusion protein comprises a DNA binding protein linked to a first, second, and third suppressor domain, wherein each of the suppressor domains is different and the fusion protein is capable of binding to a PCKS9 target nucleic acid. In some embodiments, the long-term suppressor fusion protein comprises a DNA binding protein linked to a first, second, third, and fourth suppressor domain, wherein each of the suppressor domains is different. In any of the foregoing embodiments, the fusion protein is capable of forming an RNP with a gRNA of the system that binds to a target nucleic acid when the DNA binding domain comprises a catalytically inactive CRISPR protein.

In some embodiments, the DNA binding protein comprises a TALE that can bind to but not cleave a target nucleic acid. In some embodiments, the DNA binding protein comprises a zinc finger protein modified to bind but not cleave a target nucleic acid. In some embodiments, the DNA binding protein comprises a catalytically ineffective CRISPR protein that can be complexed with a gRNA to form an RNP that can bind but not cleave a target nucleic acid. In some embodiments, the long-term suppressor fusion protein comprises a catalytically ineffective CRISPR protein sequence, a first suppressor domain (hereinafter referred to as "RD1"), a DNMT3A catalytic domain from a DNMT3A protein as a second domain (hereinafter referred to as "DNMT3A"), and a DNMT3L interaction domain from a DNMT3L protein as a third domain (hereinafter referred to as "DNMT3L"). In some embodiments, the long-term suppressor fusion protein comprises a catalytically inactive CRISPR protein sequence, RD1, DNMT3A as a second domain, DNMT3L as a third domain, and an ATRX-DNMT3-DNMT3L domain (hereinafter referred to as "ADD") from a DNMT3A protein as a fourth domain. In some embodiments, the long-term suppressor fusion protein comprises dCasX and further comprises a first and a second NLS and one or more linker peptides described herein. In some embodiments, the long-term suppressor fusion protein is capable of forming an RNP with a gRNA bound to a target nucleic acid. It has been found that when the aforementioned domains are configured in a selected orientation relative to dCasX in a long-term repressor fusion protein, the use of the aforementioned domains can lead to obvious epigenetic modification of the PCSK9 target nucleic acid when complexed with a gRNA with a targeting sequence complementary to a defined region of the PCSK9 gene, and the combination of repressor domains acts synchronously, resulting in either additive or synergistic effects on the transcriptional silencing of the target gene, depending on the configuration.

This article provides representative amino acid sequences of components used in long-term suppressor fusion protein constructs.

In some embodiments, the dCasX of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 4 to 29, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the dCasX of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 4 to 29. In some embodiments, the dCasX of the long-term suppressor fusion protein comprises a sequence of SEQ ID NO: 4, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the dCasX of the long-term suppressor fusion protein comprises the sequence of SEQ ID NO: 4.

In some embodiments, the RD1 of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 128 to 1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the RD1 of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 128 to 1726. In some embodiments, the RD1 of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 128 or SEQ ID NO: 129, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the RD1 of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 128 or SEQ ID NO: 129. In another embodiment, the first suppressor domain (RD1) of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 130 to 1726, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first suppressor domain (RD1) of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 130 to 1726. In another embodiment, the first inhibitor domain (RD1) of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 130 to 224, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first inhibitor domain (RD1) of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 130 to 224. In another embodiment, the first inhibitor domain (RD1) of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 130 to 138, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first inhibitor domain (RD1) of the long-term suppressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 130 to 138. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises the sequence of SEQ ID NO: 135, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises the sequence of SEQ ID NO: 131, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises the sequence of SEQ ID NO: 135. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises the sequence of SEQ ID NO: 130 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises the sequence of SEQ ID NO: 130. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises the sequence of SEQ ID NO: 131. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises the sequence of SEQ ID NO: 132. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises the sequence of SEQ ID NO: 133. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises a sequence of SEQ ID NO: 134. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises a sequence of SEQ ID NO: 135. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises a sequence of SEQ ID NO: 136. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises a sequence of SEQ ID NO: 137. In another embodiment, the first inhibitor domain (RD1) of the long-term inhibitor fusion protein comprises a sequence of SEQ ID NO: 138.

In some embodiments, the second inhibitor domain of the long-term inhibitor fusion protein is DNMT3A, which comprises the sequence of SEQ ID NO: 126 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the second inhibitor domain of the long-term inhibitor fusion protein comprises the sequence of SEQ ID NO: 126.

In some embodiments, the third inhibitor domain of the long-term inhibitor fusion protein is DNMT3L, which comprises the sequence of SEQ ID NO: 127 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

In some embodiments, the optional fourth inhibitory subdomain of the long-term inhibitor fusion protein is ADD, which comprises the sequence of SEQ ID NO: 125 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the optional fourth inhibitory subdomain of the long-term inhibitor fusion protein comprises the sequence of SEQ ID NO: 125. In some embodiments, the C-terminus of ADD is linked to the N-terminus of DNMT3A. It has been found that the addition of ADD to a long-term inhibitor fusion protein comprising RD1, DNMT3A and DNMT3L can greatly enhance or increase long-term inhibition and/or epigenetic modification of target nucleic acids, as well as the specificity of inhibition, compared to a long-term inhibitor fusion protein lacking ADD. Exemplary data regarding improved inhibition and specificity of long-term inhibitor fusion proteins comprising ADD are presented in the Examples and described in WO2023049742A2, which is incorporated herein by reference. In some embodiments, the fusion protein comprises one or more linker peptides selected from the group consisting of SEQ ID NOs: 98 to 124, 3278 to 3289 (exemplary sequences are shown in Tables 5 and 58) and one or more NLSs comprising a sequence selected from the group consisting of SEQ ID NOs: 30 to 97, 32998 and 3299. An exemplary configuration of a long-term suppressor fusion protein comprising ADD is presented in Figure 61.

In some embodiments, the long-term suppressor fusion protein comprises DNMT3A, DNMT3L, a DNA binding protein, and RD1 from the N-terminus to the C-terminus. In some embodiments, the long-term suppressor fusion protein comprises ADD, DNMT3A, DNMT3L, a DNA binding protein, and RD1 from the N-terminus to the C-terminus. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically ineffective CRISPR protein. In some embodiments, the long-term suppressor fusion protein comprises an NLS at the N-terminus, the C-terminus, or both. In some embodiments, the long-term suppressor fusion protein comprises one or more linkers between DNMT3A and DNMT3L, between DNMT3L and the DNA binding protein, and/or between the DNA binding protein and RD1.

In some embodiments, the long-term suppressor fusion protein comprises a DNA binding protein, RD1, DNMT3A, and DNMT3L from the N-terminus to the C-terminus. In some embodiments, the long-term suppressor fusion protein comprises a DNA binding protein, RD1 and ADD, DNMT3A, and DNMT3L from the N-terminus to the C-terminus. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically ineffective CRISPR protein. In some embodiments, the long-term suppressor fusion protein comprises an NLS at the N-terminus. In some embodiments, the long-term suppressor fusion protein comprises an NLS between RD1 and DNMT3A. In some embodiments, the long-term suppressor fusion protein comprises an NLS at the N-terminus, the C-terminus, or both. In some embodiments, the long-term suppressor fusion protein comprises one or more linkers between the N-terminal NLS and the DNA binding protein, between the DNA binding protein and RD1, between RD1 and DNMT3A or optionally ADD, and/or between DNMT3A and DNMT3A.

In some embodiments, the long-term suppressor fusion protein comprises a DNA binding protein, DNMT3A, DNMT3L, and RD1 from the N-terminus to the C-terminus. In some embodiments, the long-term suppressor fusion protein comprises a DNA binding protein, ADD, DNMT3A, DNMT3L, and RD1 from the N-terminus to the C-terminus. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically ineffective CRISPR protein. In some embodiments, the long-term suppressor fusion protein comprises an NLS at the N-terminus, the C-terminus, or both. In some embodiments, the long-term suppressor fusion protein comprises one or more linkers between the N-terminal NLS and the DNA binding protein, between the DNA binding protein and DNMT3A or ADD, if present, between DNMT3A and DNMT3L, and/or between DNMT3L and RD1.

In some embodiments, the long-term suppressor fusion protein comprises RD1, DNMT3A, DNMT3L and a DNA binding protein from the N-terminus to the C-terminus. In some embodiments, the long-term suppressor fusion protein comprises RD1, ADD, DNMT3A, DNMT3L and a DNA binding protein from the N-terminus to the C-terminus. In some embodiments, the DNA binding protein may be a zinc finger, a TALE or a catalytically ineffective CRISPR protein. In some embodiments, the long-term suppressor fusion protein comprises an NLS at the N-terminus, the C-terminus or both. In some embodiments, the long-term suppressor fusion protein comprises one or more linkers between RD1 and DNMT3A or ADD, as appropriate, between DNMT3A and DNMT3L, between DNMT3L and a DNA binding protein and/or between a DNA binding protein and a C-terminal NLS.

In some embodiments, the long-term suppressor fusion protein comprises DNMT3A, DNMT3L, RD1 and a DNA binding protein from the N-terminus to the C-terminus. In some embodiments, the long-term suppressor fusion protein comprises ADD, DNMT3A, DNMT3L, RD1 and a DNA binding protein from the N-terminus to the C-terminus. In some embodiments, the DNA binding protein may be a zinc finger, a TALE or a catalytically ineffective CRISPR protein. In some embodiments, the long-term suppressor fusion protein comprises an NLS at the N-terminus, the C-terminus or both. In some embodiments, the long-term suppressor fusion protein comprises one or more linkers between DNMT3A and DNMT3L, between DNTM3L and RD1, between RD1 and a DNA binding protein and/or between a DNA binding protein and a C-terminal NLS.

In some embodiments, the long-term suppressor fusion protein comprises DNMT3A, DNMT3L, RD1, a DNA binding protein, and a second RD1 from the N-terminus to the C-terminus. In some embodiments, the long-term suppressor fusion protein comprises ADD, DNMT3A, DNMT3L, RD1, a DNA binding protein, and a second RD1 from the N-terminus to the C-terminus. In one embodiment of the foregoing, the sequence of the second RD1 may be consistent with the first RD1. In another embodiment of the foregoing, the sequence of the second RD1 may be different from the first RD1. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically ineffective CRISPR protein. In some embodiments, the long-term suppressor fusion protein comprises an NLS at the N-terminus, the C-terminus, or both. In some embodiments, the long-term suppressor fusion protein comprises one or more linkers between DNMT3A and DNMT3L, between DNMT3L and RD1, between RD1 and the DNA binding protein, between the DNA binding protein and a second RD1, and/or between the second RD1 and the C-terminal NLS.

In some cases, the long-term suppressor fusion protein also comprises one or more NLSs. In some embodiments, the long-term suppressor fusion protein comprises the following N-terminal to C-terminal configurations: NLS-ADD-DNMT3A-DNMT3L-DNA binding protein-RD1-NLS, NLS-DNA binding protein-RD1-NLS-ADD-DNMT3A-DNMT3L, NLS-DNA binding protein-ADD-DNMT3A-DNMT3L-RD1-NLS), NLS-RD1-ADD-DNMT3A-DNMT3L-DNA binding protein-NLS, NLS-ADD-DNMT3A-DNMT3L-RD1-DNA binding protein-NLS, or NLS-ADD-DNMT3A-DNMT3L-RD1-DNA binding protein-RD1-NLS. In some embodiments, the DNA binding protein can be a zinc finger, a TALE, or a catalytically inactive CRISPR protein. In some embodiments, the long-term suppressor fusion protein is capable of binding to the PCSK9 gene and inhibiting or silencing the transcription of the PCSK9 gene.

In some embodiments of the long-term suppressor fusion protein, one or more linker peptides can be inserted between any two adjacent domains of the long-term suppressor fusion protein. In some embodiments, the long-term suppressor fusion protein comprises the following N-terminal to C-terminal configuration: NLS-ADD-DNMT3A-Linker 2-DNMT3L-Linker 1-Linker 3A-DNA binding protein-Linker 3B-RD1-NLS (Configuration 1). In some embodiments, the long-term suppressor fusion protein comprises the following N-terminus to C-terminus configuration: NLS-Linker 3A-DNA Binding Protein-Linker 3B-RD1-NLS-Linker 1-ADD-DNMT3A-Linker 2-DNMT3L (Configuration 2) In some embodiments, the long-term suppressor fusion protein comprises the following N-terminus to C-terminus configuration: NLS-Linker 3A-DNA Binding Protein-Linker 1-ADD-DNMT3A-Linker 2-DNMT3L-Linker 3B-RD1-NLS (Configuration 3). In some embodiments, the long-term suppressor fusion protein comprises the following N-terminus to C-terminus configuration: NLS-RD1-Linker 3A-ADD-DNMT3A-Linker 2-DNMT3L-Linker 1-DNA Binding Protein-Linker 3B-NLS (Configuration 4). In some embodiments, the long-term suppressor fusion protein comprises the following N-terminal to C-terminal configuration: NLS-ADD-DNMT3A-Linker 2-DNMT3L-Linker 3A-RD1-Linker 1-DNA binding protein-Linker 3B-NLS (Configuration 5). In some embodiments, the DNA binding protein in the aforementioned configuration can be a zinc finger, a TALE, or a catalytically inactive CRISPR protein. Schematic diagrams of these configurations are depicted in Figure 61. In some embodiments of LTRPs having configurations 1 to 5, the NLS can comprise a sequence selected from the group consisting of SEQ ID NOs: 30 to 97, 3298, and 3299 (Tables 3 and 4), and the linker sequence can comprise a sequence independently selected from the group consisting of: SEQ ID NOs: 98 to 124, 3278 to 3289 (representative linkers shown in Tables 5 and 58). In some embodiments, the NLS may comprise a sequence of SEQ ID NO: 30, and the linker sequence may comprise a sequence independently selected from the group consisting of SEQ ID NOs: 120 and 122 to 124. In some embodiments, the DNA binding protein may be a dCasX sequence selected from the group consisting of SEQ ID NOs: 4 to 29, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the second inhibitor domain is a DNMT3A domain comprising a sequence of SEQ ID NO: 126 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the third inhibitor is a DNMT3L domain comprising a sequence of SEQ ID NO: 127 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the fourth inhibitor is an ADD domain comprising a sequence of SEQ ID NO: 125 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the long-term inhibitor fusion protein is capable of binding to a PCSK9 gene target nucleic acid and inhibiting or silencing the PCSK9 gene target nucleic acid.

In some embodiments, the disclosure provides a system comprising a long-term inhibitor fusion protein, which comprises two RD1 domains, a second inhibitor domain, a third inhibitor domain, and a fourth inhibitor domain operably linked to a DNA binding protein, such as a zinc finger, a TALE, or a catalytically ineffective CRISPR protein. In some embodiments, the DNA binding protein comprises a catalytically ineffective CasX, which comprises a sequence selected from the group consisting of SEQ ID NO: 4 to 29 or a sequence having at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity therewith. In some embodiments, the sequences of the two RD1s are identical. In other embodiments, the two RD1s comprise different sequences. In some embodiments, the two RD1s are located at the N-terminus of the DNA binding protein. In some embodiments, two RD1s are located at the C-terminus of the DNA binding protein. In some embodiments, one RD1 is located at the N-terminus of the DNA binding protein and one RD1 is located at the C-terminus of the DNA binding protein.

In some embodiments, the long-term suppressor fusion protein comprises two RD1s. In some embodiments, the long-term suppressor fusion protein comprises the following N-terminal to C-terminal configuration: NLS-ADD-DNMT3A-Linker 2-DNMT3L-Linker 3A-a RD1a-Linker 1-DNA binding protein-Linker 3B-RD1a-Linker 4-NLS (Configuration 6a), wherein the RD1a sequence is identical (see Figure 61 for a schematic diagram of a long-term suppressor fusion protein). In some embodiments, the long-term suppressor fusion protein comprises the following N-terminal to C-terminal configuration: NLS-ADD-DNMT3A-Linker 2-DNMT3L-Linker 3A-a RD1a-Linker 1-DNA binding protein-Linker 3B-RD1b-Linker 4-NLS (Configuration 6b), wherein the RD1a sequence is different from the RD1b sequence (see Figure 61 for a schematic diagram of a long-term suppressor fusion protein). In some embodiments, the DNA binding protein can be a zinc finger, a TALE, or a catalytically ineffective CRISPR protein. In some embodiments, the long-term suppressor protein embodiments of this paragraph are capable of binding to the expression of the PCSK9 gene and inhibiting or silencing the expression of the PCSK9 gene.

In some embodiments, the disclosure provides a long-term suppressor fusion protein having configuration 6a, wherein two RD1 sequences are identical. In some embodiments of the configuration 6a long-term suppressor fusion protein, the DNA binding protein comprises a dCasX having a sequence of SEQ ID NO: 4, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the first and second copies of RD1a comprise a sequence selected from the group consisting of SEQ ID NOs: 130 to 1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto, and the second suppressor domain comprises SEQ ID NO: The first inhibitor is a DNMT3L comprising the sequence of SEQ ID NO: 127 or a sequence variant thereof having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto; The second inhibitor is a DNMT3L comprising the sequence of SEQ ID NO: 127 or a sequence variant thereof having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto; 125, or an ADD with a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the NLSs comprising a sequence independently selected from the group consisting of SEQ ID NOs: 30 to 97 (Tables 3 and 4), the L1 linker comprising the sequence of SEQ ID NO: 123, the L2 linker comprising the sequence of SEQ ID NO: 122, the L3A linker comprising the sequence of SEQ ID NO: 124, the L3B linker comprising the sequence of SEQ ID NO: 120, and the L4 linker comprising the sequence of SEQ ID NO: 3288 or SEQ ID NO: 3289. In some embodiments of long-term suppressor fusion proteins with configuration 6a, linker sequences are independently selected from the group consisting of SEQ ID NOs: 98 to 124, 3278 to 3289 (exemplary sequences shown in Tables 5 and 58). In some embodiments of long-term suppressor fusion proteins with configuration 6a, wherein the two copies of RD1 are identical, each of RD1 comprises a sequence selected from the group consisting of SEQ ID NOs: 130, 131, and 135, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% identity thereto. A schematic diagram of configuration 6a is shown in Figure 61. In some embodiments, the long-term suppressor fusion protein with configuration 6a is capable of forming RNP with the gRNA disclosed herein and is capable of binding to a gene target nucleic acid and inhibiting or silencing the gene target nucleic acid.

In some embodiments of Configuration 6b long-term suppressor fusion proteins, wherein the two RD1 sequences are identical, the DNA binding protein comprises a dCasX having a sequence of SEQ ID NO: 4, or a sequence having at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the first copy of the first suppressor domain (RD1a) comprises a sequence selected from the group consisting of SEQ ID NOs: 130 to 1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto, and the second copy of the first suppressor domain (RD1b) comprises a sequence selected from the group consisting of SEQ ID NOs: The second inhibitory subdomain is a DNMT3A comprising a sequence of SEQ ID NO: 126 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 96% identity thereto, and the third inhibitor is a DNMT3A comprising SEQ ID NO: 126 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto, the fourth inhibitor is an ADD comprising a sequence of SEQ ID NO: 125, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the NLS comprises a sequence independently selected from the group consisting of SEQ ID NOs: 30 to 97 of Tables 3 and 4, the L1 linker comprises the sequence of SEQ ID NO: 123, the L2 linker comprises SEQ ID NO: In some embodiments, the L3A linker comprises the sequence of SEQ ID NO: 122, the L3A linker comprises the sequence of SEQ ID NO: 124, the L3B linker comprises the sequence of SEQ ID NO: 120, and the L4 linker comprises the sequence of SEQ ID NO: 3288 or SEQ ID NO: 3289. In some embodiments of long-term suppressor fusion proteins having configuration 6b, the linker sequence is selected from the group consisting of SEQ ID NOs: 98 to 124, 3278 to 3289 (exemplary sequences shown in Tables 5 and 58). In some embodiments of Configuration 6b long-term suppressor fusion proteins, wherein the two copies of RD1 are different, RD1a comprises the sequence of SEQ ID NO: 130, or a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical thereto, and RD1b comprises the sequence of SEQ ID NO: 131, or a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical thereto. In some embodiments of Configuration 6b long-term suppressor fusion proteins, wherein the two copies of RD1 are different, RD1a comprises the sequence of SEQ ID NO: 130, or a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical thereto, and RD1b comprises the sequence of SEQ ID NO: 135, or a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical thereto. In some embodiments of Configuration 6b long-term suppressor fusion proteins, wherein the two copies of RD1 are different, RD1a comprises the sequence of SEQ ID NO: 131, or a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical thereto, and RD1b comprises the sequence of SEQ ID NO: 130, or a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical thereto. In some embodiments of Configuration 6b long-term suppressor fusion proteins, wherein the two copies of RD1 are different, RD1a comprises the sequence of SEQ ID NO: 131, or a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical thereto, and RD1b comprises the sequence of SEQ ID NO: 135, or a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical thereto. In some embodiments of Configuration 6b long-term suppressor fusion proteins, wherein the two copies of RD1 are different, RD1a comprises the sequence of SEQ ID NO: 135, or a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical thereto, and RD1b comprises the sequence of SEQ ID NO: 130, or a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical thereto. In some embodiments of the configuration 6b long-term suppressor fusion protein, wherein the two copies of RD1 are different, RD1a comprises the sequence of SEQ ID NO: 135, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto, and RD1b comprises the sequence of SEQ ID NO: 131, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto. In some embodiments of the configuration 6b long-term suppressor fusion protein, wherein the two copies of RD1 are different, RD1a and Rd1b are independently selected from the group consisting of sequences of SEQ ID NOs: 132 to 134 and 136 to 138. In some embodiments of the long-term suppressor fusion protein of configuration 6b, wherein the two copies of RD1 are different, RD1a and Rd1b are independently selected from the group consisting of sequences of SEQ ID NO: 130, 131 and 135. A schematic diagram of configuration 6b is shown in Figure 61. In some embodiments, the long-term suppressor fusion protein is capable of forming RNPs with the gRNA disclosed herein and is capable of binding to a gene target nucleic acid and inhibiting or silencing the gene target nucleic acid. Table 5 : Exemplary linker amino acid sequences of long-term suppressor fusion proteins Connector location Connecting Subcollections# Amino acid sequence SEQ ID NO L1 Baseline/Original GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE 123 Collection 85 ASPAAPAPASPAAPAPSAPAA 3278 Collection 43 GSGGSGGSGGSPVPSTPPTPSPSTPPTP 3279 Collection 36 PPTPSPSPVPSTPPTNSSSTPPTPS 3280 Collection 1 ASAAAPAAASAAASAPSAAAA 3281 Collection 66 AASPAAPSAPPAAASP 3282 L2 Baseline/Original SSGNSNANSRGPSFSSGLVPLSLRGSH 122 L3A Baseline/Original GGSGGG 124 Collection 85 IRAHGD 3283 Collection 43 AFPAAPAPA 3284 Collection 36 GSGNSSGSGGS 3285 Collection 1 ASAAAPAAA 3286 Collection 66 PPTP 3287 L3B Baseline/Original GGSGGGS 120 L4v1 Baseline/Original GSGS 3288 L4v2 Baseline/Original GSGSGSG 3289

In some embodiments of long-term suppressor fusion proteins comprising dCasX and, optionally, ADD and configured in Configuration 1, the long-term suppressor protein comprises a sequence selected from the group consisting of SEQ ID NOs: 22836-22855, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity thereto. In some embodiments of long-term suppressor fusion proteins comprising dCasX and, optionally, ADD and configured in Configuration 1, the long-term suppressor protein comprises a sequence selected from the group consisting of SEQ ID NOs: 22836-22855. In some embodiments of long-term suppressor fusion proteins comprising dCasX and optionally ADD and configured in Configuration 1, the long-term suppressor protein comprises a sequence selected from the group consisting of SEQ ID NOs: 22838 and 22847. In some embodiments of long-term suppressor fusion proteins comprising dCasX and optionally ADD and configured in Configuration 1, the long-term suppressor protein comprises a sequence selected from the group consisting of SEQ ID NOs: 22839 and 22848. In some embodiments of long-term suppressor fusion proteins comprising dCasX and optionally ADD and configured in Configuration 1, the long-term suppressor protein comprises a sequence selected from the group consisting of SEQ ID NOs: 22840 and 22849.

In some embodiments of the system comprising a DNA binding protein and configured in a configuration selected from the group consisting of Configuration 1, Configuration 2, Configuration 3, Configuration 4, Configuration 5, Configuration 6a and Configuration 6b, the long-term suppressor fusion protein comprises dCasX and is capable of complexing with a gRNA having a targeting sequence complementary to a PCSK9 target nucleic acid in a cell to form an RNP, and after binding the RNP to the PCSK9 target nucleic acid in the cell, the target nucleic acid is epigenetically modified and the transcription of the PCSK9 gene is inhibited. In some embodiments, the transcription of the PCSK9 gene is inhibited by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least 99%. In some embodiments, the transcriptional inhibition of the PCSK9 gene is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% or more of the cells in a cell population. In some embodiments, the transcriptional inhibition is analyzed in an in vitro assay (including a cell-based assay) when compared to untreated cells or cells treated with a comparable system comprising a non-targeting spacer. In some embodiments, transcriptional inhibition is analyzed in vivo in cells obtained from an individual to whom a long-term suppressor fusion protein and a gRNA having a targeting sequence complementary to a PCSK9 target nucleic acid of a gene in the cell are administered; in the form of a protein and a gRNA or in the form of a nucleic acid (e.g., a gRNA and an mRNA encoding a long-term suppressor fusion protein), wherein the individual is selected from the group consisting of a mouse, a rat, a pig, a non-human primate, and a human.

Optimally, PCSK9 gene inhibition results in complete inhibition of gene expression, making it impossible to detect the gene product. However, skilled artisans will appreciate that incomplete inhibition is still useful and desirable for a variety of applications. In some embodiments, when analyzed in an in vitro assay (including a cell-based assay), inhibition of transcription of the PCSK9 gene by an embodiment of the system lasts for at least about 8 hours, at least about 1 day, at least about 7 days, at least 2 weeks, at least about 3 weeks, at least about 1 month, or at least about 2 months. In some embodiments, when a composition comprising the system of the present disclosure is administered in a therapeutically effective amount, the inhibition of transcription of the PCSK9 gene by the system composition in the targeted cells of the subject lasts for at least about 7 days, at least 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months. In some embodiments, the subject is selected from the group consisting of mice, rats, pigs, non-human primates, and humans. In some embodiments, use of LTRPs of configurations 1, 4, 5, and 6 results in less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than 0.5%, or less than 0.1% off-target methylation or off-target activity in cells when used in the LTRP:gRNA systems of the embodiments.

In some embodiments, transcriptional inhibition of the PCSK9 gene in cells treated with the LTRP:gRNA system of the embodiments is heritable and stable through one or more cell divisions. In some embodiments, transcriptional inhibition is stable through 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more cell divisions. V. mRNA Compositions Encoding Long-Term Suppressor Fusion Proteins

In another aspect, the disclosure relates to messenger RNA (mRNA) compositions comprising sequences of various components and mRNA sequences of long-term inhibitor domain fusion protein constructs disclosed herein. In some embodiments, when used to express long-term inhibitor fusion proteins, mRNA compositions have utility in transcriptional inhibition and epigenetic modification of genes (e.g., PCSK9 genes). In some cases, mRNA is designed for certain delivery formulations; for example, nanoparticles, such as synthetic nanoparticles or lipid nanoparticles (LNPs). The disclosure also provides methods for designing mRNA sequences of mRNAs used in compositions. In some cases, mRNAs encoding long-term inhibitor fusion proteins can be co-formulated in nanoparticles (e.g., LNPs) with gRNAs comprising targeting sequences complementary to the sequences of PCSK9 genes. After delivery to the target cell via LNP, the long-term inhibitory subdomain fusion protein is expressed from the mRNA and can be complexed with the gRNA to form an RNP capable of binding to the PCSK9 target nucleic acid. In other cases, the mRNA encoding the long-term inhibitory subdomain fusion protein and the gRNA can be formulated in separate nanoparticles and delivered separately or as a mixture.

In some embodiments, the mRNA compositions disclosed herein have been modified to produce one or more improved characteristics relative to unmodified mRNA encoding the same long-term suppressor protein, and thus can have a positive impact on the efficacy of mRNA-based delivery. Exemplary improved characteristics of the mRNA described herein include, but are not limited to, improved expression after delivery to cells, reduced immunogenicity, increased stability, and enhanced manufacturability compared to unmodified mRNA. In some cases, the modification of the mRNA results in improved characteristics that are at least about 1.1 to about 100,000 times improved relative to the unmodified mRNA. In some embodiments, the improved characteristics of the modified mRNA are at least about 1.1 to about 10,000-fold improved, at least about 1.1 to about 1,000-fold improved, at least about 1.1 to about 500-fold improved, at least about 1.1 to about 400-fold improved, at least about 1.1 to about 300-fold improved, at least about 1.1 to about 200-fold improved, at least about 1.1 to about 100-fold improved, at least about 1.1 to about 50-fold improved, at least about 1.1 to about 40-fold improved, at least about 1.1 to about 30-fold improved, at least about 1.1 to about 20-fold improved, at least about 1.1 to about 10-fold improved, at least about 1.1 to about 15-fold improved, at least about 1.1 to about 16-fold improved, at least about 1.1 to about 17-fold improved, at least about 1.1 to about 18-fold improved, at least about 1.1 to about 19-fold improved, at least about 1.1 to about 20-fold improved, at least about 1.1 to about 21-fold improved, at least about 1.1 to about 23-fold improved, at least about 1.1 to about 24-fold improved, at least about 1.1 to about 25-fold improved, at least about 1.1 to about 26-fold improved, at least about 1.1 to about 27-fold improved, at least about 1.1 to about 28-fold improved, at least about 1.1 to about 29-fold improved, at least about 1.1 to about 30-fold improved, at least about 1.1 to about 31-fold improved, at least about 1.1 to about 31-fold improved, at least about 1.1 to about 32-fold improved, In some embodiments, the modified mRNA has an improved characteristic of at least about 10 to about 1000 times, ...500 times, an improved characteristic of at least about 10 to about 1500 times, an improved characteristic of at least about 10 to about 1500 times, an improved characteristic of at least about 10 to about 1500 times, an improved characteristic of at least about

Optimization of coding sequences and non-translated regions (UTRs) may be applicable when delivering mRNAs that encode proteins of interest, as opposed to DNA templates that will be transcribed into mRNAs. DNA templates are long-lived, can replicate, and can produce many RNA transcripts during their life cycle. For DNA templates, transcription efficiency and pre-mRNA processing are the main determinants of the amount of protein expressed. In contrast, mRNAs generally have a much shorter half-life, on the order of hours, because they are susceptible to cytoplasmic degradation and cannot produce more copies themselves. Therefore, mRNA stability and translation efficiency can be key determinants of the amount of protein expressed for mRNA-based delivery, and therefore, the specific sequences of UTRs and coding sequences that determine mRNA stability and translation efficiency can be enhanced to improve the efficacy of mRNA-based delivery. a. 5' cap

In some embodiments of the mRNA disclosed herein, the mRNA comprises a 5' cap linked to the 5' UTR of any of the embodiments described herein. In some embodiments, the 5' cap is a 7-methylguanylate cap. In some embodiments, the 5' cap comprises m7G(5')ppp(5')mAG. In other embodiments, the 5' cap comprises m7G(5')ppp(5')A or G(5')ppp(5')G. Exemplary caps are known in the art and are described, for example, in WO 2017/053297, the contents of which are incorporated herein by reference. b. 5' untranslated region (UTR)

The 5' UTR of an mRNA molecule can be a key determinant of the stability of the mRNA and how efficiently it is translated into protein. Specifically, the 5' UTR in conjunction with the 5' cap structure serves as a binding site and recruitment platform for the translation preinitiation complex as well as additional regulatory proteins that can have a positive or negative effect on translation. Structures within the 5' UTR can enhance translation by recruiting initiation factors or other proteins or RNA factors, reduce translation by physically blocking ribosome binding and scanning, and promote mRNA stability by affecting both hydrolysis and nuclease digestion.

Exemplary 5'UTR sequences used in the mRNA of the present disclosure are provided in Table 6. Table 6 lists RNA sequences and RNA sequences with N1-methylpseudouridine substituted for uridine. Table 6 : 5'UTR sequences SEQ ID NO Nucleic acid sequence * describe 22787 AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 5' UTR RNA sequence 3300 AAAmΨAAGAGAGAAAAGAAGAGmΨAAGAAGAAAmΨAmΨAAGA RNA sequence with N1-methylpseudouridine substitution in 5'UTR *『mψ』=N1-methyl-pseudouridine

In some embodiments, the 5'UTR comprises a sequence of SEQ ID NO: 22787 or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, the 5'UTR comprises a sequence of SEQ ID NO: 22787. In some embodiments, the 5'UTR consists of a sequence of SEQ ID NO: 22787. In some embodiments, the 5'UTR comprises a sequence of SEQ ID NO: 3300 or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, the 5'UTR comprises a sequence of SEQ ID NO: 3300. In some embodiments, the 5'UTR consists of a sequence of SEQ ID NO: 3300. c. 3'UTR

The 3'UTR sequence can affect mRNA stability and translation efficiency, and therefore can determine subcellular localization and tissue-specific expression. Factors affecting these properties include microRNA binding sites, AU-rich elements that recruit a series of RNA-binding proteins, Pumilio binding elements, and other binding sites for RNA-binding proteins. Although many of these interactions with the 3'UTR are known to negatively affect stability or expression, some can enhance translation. The effect of the 3'UTR sequence can be highly cell-type specific due to the differential expression of microRNAs and RNA-binding proteins, which provides the opportunity to engineer tissue-specific expression of therapeutic mRNAs. In some embodiments, the 3'UTR used for the mRNA disclosed herein is a mouse 3'UTR. In some embodiments, the 3'UTR is the mouse HBA gene 3'UTR of Table 7.

Exemplary 3'UTR sequences of the present disclosure are provided in Table 7. Table 7 lists RNA sequences and RNA sequences with N1-methylpseudouridine substituted for uridine. Table 7 : 3'UTR sequences SEQ ID NO Nucleic acid sequence* describe 3125 GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG 3' UTR RNA sequence 3310 GCmΨGCCmΨmΨCmΨGCGGGGCmΨmΨGCCmΨmΨCmΨGGCCAmΨGCCCmΨmΨCmΨmΨCmΨCmΨ CCCmΨmΨGCACCmΨGmΨACCmΨCmΨmΨGGmΨCmΨmΨmΨGAAmΨAAAGCCmΨGAGmΨAGGAAG RNA sequence with N1-methylpseudouridine substitution in 3'UTR *『mψ』=N1-methyl-pseudouridine

In some embodiments, the 3'UTR comprises a sequence of SEQ ID NO: 3125, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, the 3'UTR comprises a sequence of SEQ ID NO: 3125. In some embodiments, the 3'UTR consists of a sequence of SEQ ID NO: 3125. In some embodiments, the 3'UTR comprises a sequence of SEQ ID NO: 3310, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, the 3'UTR comprises a sequence of SEQ ID NO: 3310. In some embodiments, the 3'UTR consists of a sequence of SEQ ID NO: 3310. d. Polyadenylation sequence

Including a 3' poly(A) tail in the mRNA sequence can contribute to the stability and translation efficiency of the mRNA. In general, longer poly(A) tails are associated with increased mRNA stability, thereby allowing its translation and promoting high protein expression.

In some embodiments, the mRNA of the present disclosure comprises a poly(A) sequence having at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides. In some embodiments, the poly(A) sequence of the mRNA of the present disclosure comprises 80 adenine nucleotides. In some embodiments, the poly(A) sequence comprises the nucleic acid sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 3057). In some embodiments, the poly(A) sequence comprises the nucleic acid sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 3307). e. Sequence modification of mRNA

In some embodiments, the mRNA sequences of the present disclosure are modified by codon optimization of the sequence encoding the long-term suppressor protein using one or more parameters to enhance expression in the target cell. Non-limiting examples of such parameters include codon usage in human host cells (e.g., using the codon adaptation index (CAI)), minimizing rare codons, using a codon usage table derived from a biological agent intended for use as a therapeutic, using an mRNA stability index, or reducing GC content. Methods for codon optimization and codon usage in various organisms are known in the art. See, e.g., www.genscript.com/tools/codon-frequency-table.

In some embodiments, provided herein are mRNA sequences for long-term suppressor protein constructs that are codon-optimized for expression in human cells. In other cases, various naturally occurring or modified nucleosides may be used to generate modified mRNAs according to the present disclosure. In some embodiments, the mRNA is or comprises the following: natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deoxythymidine ... In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. Non-standard nucleotide residues may include, for example, 5-methyl-cytidine ("5 mC"), pseudouridine ("ψU"), and/or 2-thio-uridine ("2sU"). In a specific embodiment, one or more or all uridine residues of the mRNA disclosed herein are replaced with 1-methyl-pseudouridine. In some embodiments, all uridine residues of the mRNA disclosed herein are replaced with 1-methyl-pseudouridine. For discussion of such residues and their incorporation into mRNA, see, for example, U.S. Patent No. 8,278,036 or WO2011012316, which are incorporated herein by reference. In some embodiments, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uridine nucleosides of the mRNA sequence are replaced with N1-methylpseudouridine.

In some embodiments, modification of the mRNA by codon optimization and/or substitution of modified nucleotides in the sequence results in improved characteristics that are at least about 1.1 to about 100,000 fold improved relative to the unmodified mRNA. In some embodiments, the improved characteristics of the modified mRNA are at least about 1.1 to about 10,000-fold improved, at least about 1.1 to about 1,000-fold improved, at least about 1.1 to about 500-fold improved, at least about 1.1 to about 400-fold improved, at least about 1.1 to about 300-fold improved, at least about 1.1 to about 200-fold improved, at least about 1.1 to about 100-fold improved, at least about 1.1 to about 50-fold improved, at least about 1.1 to about 40-fold improved, at least about 1.1 to about 30-fold improved, at least about 1.1 to about 20-fold improved, at least about 1.1 to about 10-fold improved, at least about 1.1 to about 15-fold improved, at least about 1.1 to about 16-fold improved, at least about 1.1 to about 17-fold improved, at least about 1.1 to about 18-fold improved, at least about 1.1 to about 19-fold improved, at least about 1.1 to about 20-fold improved, at least about 1.1 to about 21-fold improved, at least about 1.1 to about 23-fold improved, at least about 1.1 to about 24-fold improved, at least about 1.1 to about 25-fold improved, at least about 1.1 to about 26-fold improved, at least about 1.1 to about 27-fold improved, at least about 1.1 to about 28-fold improved, at least about 1.1 to about 29-fold improved, at least about 1.1 to about 30-fold improved, at least about 1.1 to about 31-fold improved, at least about 1.1 to about 31-fold improved, at least about 1.1 to about 32-fold improved, 9-fold, at least about 1.1 to about 8-fold, at least about 1.1 to about 7-fold, at least about 1.1 to about 6-fold, at least about 1.1 to about 5-fold, at least about 1.1 to about 4-fold, at least about 1.1 to about 3-fold, at least about 1.1 to about 2-fold, at least about 1.1 to about 1.5-fold, at least about 1.5 to about 3-fold, at least about 1.5 to about 4-fold, at least about 1.5 to about 5-fold, at least about 1.5 to about 10-fold, at least about 5 to about 10-fold, at least about 10 to about 20-fold, at least about 10 to about 30-fold, at least about 10 to about 50-fold, or at least about 10 to about 100-fold. In some embodiments, the improved characteristics of the modified mRNA are at least about 10 to about 1000-fold improved relative to the unmodified mRNA. f. LTRP mRNA component sequences

The present disclosure provides mRNAs comprising sequences encoding components used in the long-term suppressor fusion proteins disclosed herein. In some embodiments, the mRNA comprises sequences encoding DNA binding proteins, including TALEs, ZFs, and catalytically inactive CRISPR proteins. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 3274 encoding dCasX 515 (SEQ ID NO: 6), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 3276 encoding dCasX 812 (SEQ ID NO: 29), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 3275 encoding dCasX 491 (SEQ ID NO: 4), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 22736 encoding dCasX676 (SEQ ID NO: 28), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

In some embodiments, the sequence of the mRNA encoding dCasX 491 (SEQ ID NO: 4) has one or more or all uridines in a pseudouridine nucleoside substitution sequence (SEQ ID NO: 22729). In some embodiments, the sequence of the mRNA encoding dCasX 515 (SEQ ID NO: 6) has one or more or all uridines in a pseudouridine nucleoside substitution sequence (SEQ ID NO: 22728). In some embodiments, the sequence of the mRNA encoding dCasX 676 (SEQ ID NO: 28) has one or more or all uridines in a pseudouridine nucleoside substitution sequence. In some embodiments, the sequence of the mRNA encoding dCasX 812 (SEQ ID NO: 29) has one or more or all uridines in a pseudouridine nucleoside substitution sequence (SEQ ID NO: 22730 or 22731).

In some embodiments, the disclosure provides mRNA sequences encoding RD1 domains. In some embodiments, the sequence of the mRNA encoding RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3120, 22804, and 3334 to 6527 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity therewith. In some embodiments, the sequence of the mRNA encoding RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3120, 22804, and 3334 to 6527. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence selected from the group consisting of SEQ ID NOs: 3334 to 3342 and 4931 to 4939 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity thereto. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence selected from the group consisting of SEQ ID NOs: 3334 to 3342 and 4931 to 4939. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3334 or SEQ ID NO: 4931 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity thereto. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3334 or SEQ ID NO: 4931. In another embodiment, the mRNA sequence encoding RD1 comprises the sequence of SEQ ID NO: 3335 or SEQ ID NO: 4932 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity thereto. In another embodiment, the mRNA sequence encoding RD1 comprises the sequence of SEQ ID NO: 3335 or SEQ ID NO: 4932. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3336 or SEQ ID NO: 4933 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity thereto. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3336 or SEQ ID NO: 4933. In another embodiment, the mRNA sequence encoding RD1 comprises the sequence of SEQ ID NO: 3337 or SEQ ID NO: 4934, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity thereto. In another embodiment, the mRNA sequence encoding RD1 comprises the sequence of SEQ ID NO: 3337 or SEQ ID NO: 4934. In another embodiment, the mRNA sequence encoding RD1 comprises the sequence of SEQ ID NO: 3338 or SEQ ID NO: 4935, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity thereto. In another embodiment, the mRNA sequence encoding RD1 comprises the sequence of SEQ ID NO: 3338 or SEQ ID NO: 4935. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3339 or SEQ ID NO: 4936, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity thereto. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3339 or SEQ ID NO: 4936. In another embodiment, the mRNA sequence encoding RD1 comprises the sequence of SEQ ID NO: 3340 or SEQ ID NO: 4937 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity thereto. In another embodiment, the mRNA sequence encoding RD1 comprises the sequence of SEQ ID NO: 3340 or SEQ ID NO: 4937. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3341 or SEQ ID NO: 4938, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity thereto. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3341 or SEQ ID NO: 4938.

In some embodiments, the mRNA comprises a sequence encoding a second inhibitory subdomain. In some embodiments, the second inhibitory subdomain comprises DNMT3A. In some embodiments, the mRNA sequence encoding DNMT3A comprises a sequence of SEQ ID NO: 3128 or SEQ ID NO: 3331 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the mRNA sequence encoding DNMT3A comprises a sequence of SEQ ID NO: 3128 or SEQ ID NO: 3331.

In some embodiments, the mRNA comprises a sequence encoding a third inhibitory subdomain. In some embodiments, the third inhibitory subdomain comprises DNMT3L. In some embodiments, the mRNA sequence encoding DNMT3L comprises a sequence of SEQ ID NO: 3119 or SEQ ID NO: 3332 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the sequence of the mRNA encoding DNMT3L comprises a sequence of SEQ ID NO: 3119 or SEQ ID NO: 3332.

In some embodiments, the mRNA comprises a sequence encoding a fourth inhibitory subdomain. In some embodiments, the fourth inhibitory subdomain comprises ADD. In some embodiments, the sequence of the mRNA encoding ADD comprises a sequence of SEQ ID NO: 3296 or SEQ ID NO: 22726 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the sequence of the mRNA encoding ADD comprises a sequence of SEQ ID NO: 3296 or SEQ ID NO: 22726.

In some embodiments, the mRNA of the present disclosure comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises GCCACCAUGG (SEQ ID NO: 22805). In some embodiments, the mRNA of the present disclosure comprises a Kozak sequence and two bases upstream of the NLS (for producing methionine and alanine upstream of the NLS). In some embodiments, the mRNA comprises the sequence GCCACCAUGGCC (SEQ ID NO: 22806) between the 5'UTR and the sequence encoding the NLS.

In another embodiment, the mRNA comprises an NLS. In some embodiments, the sequence encoding the NLS comprises a sequence selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807 or a sequence having at least about 70%, at least about 80%, or at least about 90% identity thereto. In another embodiment, the mRNA comprises a sequence selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807. In another embodiment (e.g., those embodiments in which the long-term inhibitor protein comprises more than one NLS), the mRNA comprises two or more sequences independently selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807.

In some embodiments, the mRNA comprises a sequence encoding a linker of a long-term suppressor fusion protein. In some embodiments, the sequence encoding the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 3312 to 3327 and 22808 to 22828, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% identity thereto. In some embodiments, the sequence encoding the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 3312 to 3327 and 22808 to 22828. In some embodiments (e.g., those embodiments in which the long-term suppressor fusion protein comprises more than one linker), the sequence encoding the linker is independently selected from the group consisting of SEQ ID NOs: 3312 to 3327 and 22808 to 22828, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% identity thereto.

In some embodiments, the mRNA comprises a sequence encoding a long-term suppressor fusion protein of a configuration provided herein. In some embodiments, the mRNA encodes a long-term suppressor fusion protein of configuration 1, and the mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 6529 to 8134, 9741 to 11347, 14628 to 16233, and 17841 to 19446, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 6529 to 8134, 9741 to 11347, 14628 to 16233, and 17841 to 19446.

In some embodiments, the mRNA comprises a sequence encoding a long-term suppressor fusion protein of configuration 1 and comprises a sequence encoding RD1, wherein the RD1 comprises the sequence of SEQ ID NO: 130. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6531, 9744, 14630 or 17843, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6531, 9744, 14630 or 17843. In some embodiments, the mRNA comprises a sequence encoding a long-term suppressor fusion protein of configuration 1 and comprises a sequence encoding RD1 of SEQ ID NO: 131. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6529, 9742, 14628 or 17841, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6529, 9742, 14628 or 17841. In some embodiments, the mRNA comprises a sequence encoding a long-term suppressor fusion protein of configuration 1, and the mRNA comprises a sequence encoding RD1 of SEQ ID NO: 132. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6535, 9748, 14634 or 17847, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6535, 9748, 14634 or 17847. In some embodiments, the mRNA comprises a sequence encoding a long-term suppressor fusion protein of configuration 1 and comprises a sequence encoding RD1 of SEQ ID NO: 133. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6536, 9749, 14635 or 17848, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6536, 9749, 14635 or 17848. In some embodiments, the mRNA comprises a sequence encoding a long-term suppressor fusion protein of configuration 1, and the mRNA comprises a sequence encoding RD1 of SEQ ID NO: 134. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6534, 9747, 14633 or 17846, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6534, 9747, 14633 or 17846. In some embodiments, the mRNA comprises a sequence encoding a long-term suppressor fusion protein of configuration 1 and comprises a sequence encoding RD1 of SEQ ID NO: 135. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6530, 9743, 14629 or 17842, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6530, 9743, 14629 or 17842. In some embodiments, the mRNA comprises a sequence encoding a long-term suppressor fusion protein of configuration 1 and comprises a sequence encoding RD1 of SEQ ID NO: 136. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6537, 9750, 14636 or 17849, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6537, 9750, 14636 or 17849. In some embodiments, the mRNA comprises a sequence encoding a long-term suppressor fusion protein of configuration 1 and comprises a sequence encoding RD1 of SEQ ID NO: 137. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6533, 9746, 14632 or 17845, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6533, 9746, 14632 or 17845. In some embodiments, the mRNA comprises a sequence encoding a long-term suppressor fusion protein of configuration 1 and comprises a sequence encoding RD1 of SEQ ID NO: 138. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6532, 9745, 14631 or 17844, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6532, 9745, 14631 or 17844.

The present disclosure provides an mRNA encoding a long-term suppressor fusion protein of configuration 5. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 8135 to 9740, 11348 to 12953, 16234 to 17839, and 19447 to 21052, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 8135 to 9740, 11348 to 12953, 16234 to 17839, and 19447 to 21052.

The present disclosure provides an mRNA encoding a long-term suppressor fusion protein of configuration 6a, wherein the encoded RD1 is identical. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NO: 12954 to 14553 and 21053 to 22652 or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NO: 12954 to 14553 and 21053 to 22652.

The present disclosure provides a mRNA encoding a long-term suppressor fusion protein of configuration 6b, wherein the encoded RD1 is different. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NO: 14554 to 14626 and 22653 to 22725 or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity therewith. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NO: 14554 to 14626 and 22653 to 22725.

In some embodiments of the mRNA encoding the long-term suppressor fusion protein of the LTRP:gRNA system disclosed herein, after the system is delivered to the targeted cell, the mRNA is expressed and the long-term suppressor fusion protein is able to complex with the gRNA and bind to the target DNA, resulting in inhibition or silencing of transcription of the PCSK9 gene in the cell. VI. Guide Nucleic Acid of the System

In another aspect, the present invention relates to a specifically designed guide RNA (gRNA) comprising a scaffold and a linked targeting sequence that is complementary to (and therefore capable of hybridizing with) a target nucleic acid sequence of a PCSK9 gene. The gRNA described herein can be used with long-term suppressor proteins and systems comprising the same to inhibit transcription of PCSK9 target nucleic acids in eukaryotic cells. As used herein, the term "gRNA" encompasses naturally occurring molecules and gRNA variants, including chimeric gRNA variants comprising domains from different gRNAs. The gRNA disclosed herein comprises a scaffold and a targeting sequence linked to the 3' end of the scaffold that is complementary to a target nucleic acid of a cell.

In some embodiments, a system comprising mRNA encoding a long-term suppressor fusion protein comprises a dCasX protein and one or more gRNAs, which together with the gRNA form a ribonucleoprotein (RNP) complex that can target and bind to a specific location in a target nucleic acid sequence of a cell after the dCasX protein is expressed in a transfected cell. The gRNA provides target specificity to the complex by including a targeting sequence (or "spacer") having a nucleotide sequence that is complementary to the sequence of the target nucleic acid sequence, while the long-term suppressor fusion protein of the system provides site-specific activities, such as binding and inhibition of a target gene, which is guided to the target site within the target nucleic acid sequence (e.g., stabilized at the target site) by virtue of the binding of the target nucleic acid sequence to the gRNA.

Examples of gRNAs and formulations of mRNAs and gRNAs for inhibition and/or epigenetic modification of PCSK9 target nucleic acids are described below. a. Reference gRNAs and gRNA variants

As used herein, "reference gRNA" refers to a CRISPR guide RNA comprising the wild-type sequence of a naturally occurring gRNA. In some embodiments, the gRNA scaffold of the present disclosure may be subjected to one or more mutation induction methods, such as those described in WO2023235818A2, WO2022120095A1, and WO2020247882A1 (incorporated herein by reference), which may include deep mutation evolution (DME), deep mutation scanning (DMS), error-prone PCR, cassette mutation induction, random mutation induction, staggered extension PCR, gene shuffling, domain swapping, or chemical modification to generate one or more gRNA variants whose properties are enhanced or altered relative to the modified gRNA scaffold. The activity of the gRNA scaffold from which the gRNA variants are derived can be used as a benchmark to compare the activities of the gRNA variants, thereby measuring the improvement of the function or other characteristics of the gRNA scaffold.

Table 8 provides sequences of reference gRNA tracr and scaffold sequences. In some embodiments, the disclosure provides gRNA variants, wherein the gRNA has a scaffold comprising a sequence having one or more nucleotide modifications relative to the reference gRNA sequence of any one of SEQ ID NOs: 1731 to 1743 of Table 8. Table 8 : Reference gRNA tracr and scaffold sequences SEQ ID NO. Nucleotide sequence 1731 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCGAUAAGUAAAACGCAUCAAAG 1732 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 1733 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGA 1734 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGG 1735 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGA 1736 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGG 1737 GUUUACACACUCCCUCUCAUAGGGU 1738 GUUUACACACUCCCUCUCAUGAGGU 1739 UUUUACAUACCCCCUCUCAUGGGAU 1740 GUUUACACACUCCCUCUCAUGGGGG 1741 CCAGCGACUAUGUCGUAUGG 1742 GCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGC 1743 GGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA b. gRNA domain and its function

The gRNA of the disclosed system comprises two segments: a targeting sequence and a protein binding segment. The targeting segment of the gRNA includes a nucleotide sequence (interchangeably referred to as a spacer, a targeter, or a targeting sequence) that complements (and thus hybridizes with) a specific sequence (target site) within a target nucleic acid sequence (e.g., a double-stranded target DNA, a target ssRNA, a strand of a target ssDNA, etc.), which is described more fully below. The targeting sequence of the gRNA is capable of binding to a target nucleic acid sequence (including a coding sequence, a complementary sequence of a coding sequence, a non-coding sequence in the context of the present disclosure) and to an auxiliary element. The protein binding segment (or "activator" or "protein binding sequence") of the gRNA interacts with (e.g., binds to) the dCasX protein into a complex, thereby forming an RNP (described more fully below). As used herein, "scaffold" refers to all parts used for guiding except the targeting sequence, which includes several regions, which are described more fully below. The properties and characteristics of CasX gRNA (wild type and variants) are described in WO2020247882A1, US20220220508A1 and WO2022120095A1, which are incorporated herein by reference.

With reference to gRNA, gRNA exists naturally as a double-guide RNA (dgRNA), wherein the targeting sub-portion and the activating factor portion each have a duplex-forming segment, and the targeting sub-portion and the activating factor portion are complementary to each other and hybridize with each other to form a double-stranded duplex (dsRNA duplex for gRNA). The term "targeter" or "targeter RNA" used herein refers to the crRNA-like molecule of the CasX double-guide RNA (crRNA: "CRISPR RNA") (and therefore refers to the crRNA-like molecule of the CasX single-guide RNA when the "activating factor" and the "targeter" are linked together (e.g., by inserting nucleotides). The crRNA has a 5' region that is attached to the tracrRNA, followed by the nucleotides of the targeting sequence. For the gRNA used in the system of the present disclosure, the scaffold is designed so that the activator portion and the target portion are covalently linked to each other (rather than hybridized to each other) and comprise a single molecule, and may be referred to as a "single-molecule gRNA", "single guide RNA", "single-molecule guide RNA", "one-molecule guide RNA" or "sgRNA". The gRNA variants of the present disclosure used in the system are all single molecule forms.

In general, the assembled gRNA of the present disclosure comprises different structured regions or domains: RNA triple helix, scaffold stem loop, extended stem loop, pseudoknot and targeting sequence, in the embodiments of the present disclosure, the targeting sequence is complementary to the sequence of the target nucleic acid and is located at the 3' end of the gRNA. The RNA triple helix, scaffold stem loop, pseudoknot and extended stem loop together with the unstructured triplet loop bridging the triplet portion are referred to as the "scaffold" of the gRNA. In some cases, the scaffold stem further comprises a bubble. In other cases, the scaffold further comprises a triple helix loop region. In other cases, the scaffold further comprises a 5' unstructured region. In some embodiments, a gRNA scaffold of the present disclosure for use in a LTRP:gRNA system comprises a scaffold stem loop having a sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1822) or a sequence having at least 1, 2, 3, 4, or 5 mismatches therewith.

Each of the structured domains contributes to establishing the overall RNA fold of the guide and retaining the function of the guide; in particular, the ability to properly complex with the dCasX protein. For example, the guide scaffold stem interacts with the helical I domain of the dCasX protein, while residues within the triple helix, triple helix loop, and pseudostem interact with the OBD of the dCasX protein. Together, these interactions confer the ability of the guide to bind to the LTRP and form a stable RNP with the LTRP, while the spacer (or targeting sequence) guides and defines the specificity of the RNP to bind to a particular sequence of DNA.

Site-specific binding of the dCasX protein of the LTRP to a target nucleic acid sequence (e.g., genomic DNA) can occur at one or more positions (e.g., a sequence of a PCSK9 target nucleic acid) determined by base pairing complementarity between the targeting sequence of the gRNA and the target nucleic acid sequence. Thus, for example, the gRNA of the present disclosure has a sequence that is complementary to a target nucleic acid adjacent to a sequence complementary to a TC protospacer adjacent motif (PAM) motif or a PAM sequence (e.g., ATC, CTC, GTC, or TTC) and can therefore hybridize with the target nucleic acid. Because the targeting sequence of the guide sequence hybridizes with the sequence of the target nucleic acid sequence, the targeting sequence can be modified by the user to hybridize with a specific target nucleic acid sequence, as long as the position of the PAM sequence is taken into account. In some embodiments, to design a targeting sequence, the target nucleic acid comprises a PAM sequence located 5' of the targeting sequence, the PAM sequence having at least one single nucleotide separating the PAM from the first nucleotide in the target nucleic acid (which is complementary to the first nucleotide of the targeting sequence). This feature distinguishes the system described herein from the Cas9 system and enables the system disclosed herein to modify different positions in a DNA sequence when compared to the Cas9 system. In some embodiments, the PAM is located on a non-targeting strand of the target region, i.e., on a strand complementary to the target nucleic acid. In some embodiments, the targeting sequence of the gRNA is complementary to a PCSK9 target nucleic acid sequence one nucleotide away from the ATC PAM sequence. In some embodiments, the targeting sequence of the gRNA is complementary to a PCSK9 target nucleic acid sequence one nucleotide away from the CTC PAM sequence. In some embodiments, the targeting sequence of gRNA is complementary to the PCSK9 target nucleic acid sequence one nucleotide away from the GTC PAM sequence. In some embodiments, the targeting sequence of gRNA is complementary to the PCSK9 target nucleic acid sequence one nucleotide away from the TTC PAM sequence. By selecting the targeting sequence of gRNA, the LTRP:gRNA system described herein can be used to inhibit the defined region of the target nucleic acid sequence or to group the sequence of specific positions in the target nucleic acid together. In some embodiments, the targeting sequence of gRNA has 15 to 22 consecutive nucleotides. In some embodiments, the targeting sequence has 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 21 consecutive nucleotides. In some embodiments, the targeting sequence consists of 20 consecutive nucleotides. In some embodiments, the targeting sequence consists of 19 consecutive nucleotides. In some embodiments, the targeting sequence consists of 18 consecutive nucleotides. In some embodiments, the targeting sequence consists of 17 consecutive nucleotides. In some embodiments, the targeting sequence consists of 16 consecutive nucleotides. In some embodiments, the targeting sequence consists of 15 consecutive nucleotides. By selecting the targeting sequence of the gRNA, the LTRP:gRNA system described herein can be used to inhibit and/or epigenetically modify a limited region of the PCSK9 target nucleic acid sequence.

The gene suppressor system disclosed herein can be designed to target the PCSK9 gene or any region of the PCSK9 gene region or any region near the gene or gene region for which transcriptional inhibition is sought. When the entire gene is to be inhibited, the present invention contemplates designing a guide with a targeting sequence complementary to a sequence covering or near the transcription start site (TSS). Depending on the promoter sequence and the starting substrate concentration, TSS selection occurs at different positions within the promoter region. The core promoter serves as a binding platform for the transcriptional machinery, which includes Pol II and its associated general transcription factor (GTF) (Haberle, V. et al. Eukaryotic core promoters and the functional basis of transcription initiation (Nat Rev Mol Cell Biol. 19(10):621 (2018)). It has been proposed that the variability in TSS selection involves DNA "scrunching" and "de-scrunching", which are characterized by: (i) In some embodiments, the target nucleic acid sequence bound by the RNP of the LTRP:gRNA system is within 1.5 kilobases (kb) of the transcription start site (TSS) in the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by the RNP of the system is within 20 base pairs (bp), 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb or 1.5 kb upstream of the TSS of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by the RNP of the system is within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb or 1.5 kb downstream of the TSS of the PCSK9 gene. bp, 500 bp, 1 kb or 1.5 kb. In some embodiments, the target nucleic acid sequence bound by the RNP of the system is within 1.5 kb upstream to 1.5 downstream, 1 kb upstream to 1 kb downstream, 500 bp upstream to 500 bp downstream, or 300 bp upstream to 300 bp downstream, or 100 bp upstream to 100 bp downstream of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by the RNP of the system is within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb or 1.5 kb of the enhancer of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by the RNP of the system is within 1 kb of the 3' to 5' non-translated region of the PCSK9 gene. kb. In other embodiments, the target nucleic acid sequence bound by the RNP of the system is within the open reading frame of the PCSK9 gene, including introns (if present). In some embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of the exons of the PCSK9 gene. In a specific embodiment, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of exon 1 of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of the introns of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of the intron-exon junction of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of the regulatory element of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of the intergenic region of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of the junction of the exons, introns and/or regulatory elements of the PCSK9 gene. In those cases where the targeting sequence is complementary to the sequence of the regulatory element, these regulatory elements include but are not limited to promoter regions, enhancer regions, intergenic regions, 5' non-translational regions (5'UTR), 3' non-translational regions (3'UTR), conservative elements, and regions containing cis-regulatory elements. The promoter region is intended to cover nucleotides within 5 kb of the start point of the coding sequence, or in the case of a gene enhancer element or a conservative element, it can be thousands of bp, hundreds of thousands of bp, or even millions of bp away from the coding sequence of the PCSK9 gene. In the foregoing, the target is one in which the target's coding PCSK9 gene is intended to be inhibited so that the PCSK9 gene product is not expressed or expressed at a lower level in the cell. In some embodiments, after the RNP of the system disclosed herein binds to the binding site of the target nucleic acid, the system can inhibit the transcription of the PCSK9 gene at the 5' binding site of the RNP. In other embodiments, after the RNP of the system binds to the binding site of the target nucleic acid, the system can inhibit the transcription of the PCSK9 gene at the 3' binding site of the RNP.

In some embodiments, the target nucleic acid comprises a PAM sequence located 5' of the targeting sequence, the PAM sequence having at least one single nucleotide separating the PAM from the first nucleotide of the targeting sequence. In some embodiments, the PAM is located on the non-targeting strand of the target region, i.e., the strand complementary to the target nucleic acid, and as shown in Table 9 below, which represents the targeting sequence of the PCSK9 target nucleic acid for attachment to the gRNA scaffold of the present disclosure; for example, gRNA 174 (SEQ ID NO: 1744), 235 (SEQ ID NO: 1745), 316 (SEQ ID NO: 1746), or a chemically modified version. In some embodiments, the PAM sequence is TTC.

In some embodiments, the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1824-2544, 2672, 2675, 2694, and 2714, as described in Table 9. In some embodiments, the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714. In some embodiments, the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1834, 1849, 1853, 1855-1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875. In some embodiments, the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NO: 1855, 1867, and 1869. In a specific embodiment, the targeting sequence of the gRNA consists of the sequence of SEQ ID NO: 1855. In another specific embodiment, the targeting sequence of the gRNA consists of the sequence of SEQ ID NO: 1867. In another specific embodiment, the targeting sequence of the gRNA consists of the sequence of SEQ ID NO: 1869. In some embodiments, the aforementioned targeting sequence is linked to the gRNA scaffold of gRNA 174 (SEQ ID NO: 1744), 235 (SEQ ID NO: 1745) or 316 (SEQ ID NO: 1746) or a chemically modified version thereof. surface 9 : Using long-term suppressor fusion proteins PCSK9 Specific targeting sequence Targeting sequence RNA-seq SEQ ID PAM Sequence TG-06-342 AAUUACAGGCAACAGGAAGG 1824 TTC TG-06-343 CCCCAUGUAAGAGAGGAAGU 1825 TTC TG-06-344 CAGUUUCUGCCUCGCCGCGG 1826 TTC TG-06-345 GCCUCGCCGCGGCACAGGUG 1827 TTC TG-06-346 CCCACCUGUGCCGCGGCGAG 1828 TTC TG-06-347 CUCCUUCACCCACCUGUGCC 1829 TTC TG-06-348 AGGCAUUCACUCCUUCACCC 1830 TTC TG-06-349 CUGUGCCUGGUGCAGUUCCC 1831 TTC TG-06-350 GUGUCAUAAAGAAAUUGCCU 1832 TTC TG-06-351 UUAUGACACAGAACUCAUGC 1833 TTC TG-06-001 GAGGAGGACGGCCUGGCCGA 1834 TTC TG-06-002 ACCGCUGCGCCAAGGUGCGG 1835 TTC TG-06-004 GCCAGGCCGUCCUCCUCGGA 1836 TTC TG-06-005 GUGCUCGGGUGCUUCGGCCA 1837 TTC TG-06-117 ACUUUGUUUGCAAAGACCUC 1838 TTC TG-06-118 GAGUGAAAUGGCCUGCUCUG 1839 TTC TG-06-119 GAGCAGGCCAUUUCACUCGG 1840 TTC TG-06-120 CUCGGAAUCUGCUGUGCAUC 1841 TTC TG-06-121 GGAAGGGCUGUCGAUACUGG 1842 TTC TG-06-123 UCCCAGUAUCGACAGCCCUU 1843 TTC TG-06-124 CAGUAUCGACAGCCCUUCCA 1844 TTC TG-06-125 AGAAAGAGCAAGCCUCAUGU 1845 TTC TG-06-128 AGAAAUCAACUGGACAAGCA 1846 TTC TG-06-131 UGAACAUGGUGUGUAAAAGG 1847 TTC TG-06-132 AGAAGAUUCAAUUUGCAAAG 1848 TTC TG-06-133 AUGGUAGGCACAAGCUCAGC 1849 TTC TG-06-134 GAAUUCUAUGGUAGGCACAA 1850 TTC TG-06-135 GGAAAGCUGAGCUUGUGCCU 1851 TTC TG-06-138 AGGGAUUUAUACUACAAAGA 1852 TTC TG-06-139 AGGAGCAGCUAGUUGGUAAG 1853 TTC TG-06-140 AAACUUAGCCUGGACCCCCU 1854 TTC TG-06-141 ACUGGCCUUAACCUGGCAGC 1855 TTC TG-06-142 UUCCACUGGCCUUAACCUGG 1856 TTC TG-06-143 GAAUCAAUCCUACUGUGGAC 1857 TTC TG-06-144 GUGGGCAGCGAGGAGUCCAC 1858 TTC TG-06-145 UGGGUCCACCUUGUCUCCUG 1859 TTC TG-06-146 GAAGUCUCACUGGUCAGCAG 1860 TTC TG-06-147 GUGUUUCCUGGGUCCACCUU 1861 TTC TG-06-149 AGCCCAGUUAGGAUUUGGGA 1862 TTC TG-06-150 UCCCUCUGCGCGUAAUCUGA 1863 TTC TG-06-151 CUCUGCGCGUAAUCUGACGC 1864 TTC TG-06-152 GCCUCGCCCUCCCCAAACAG 1865 TTC TG-06-153 GUUAAUGUUUAAUCAGAUAG 1866 TTC TG-06-154 AGGGUGUGGGUGCUUGACGC 1867 TTC TG-06-155 GCAGCGACGUCGAGGCGCUC 1868 TTC TG-06-157 GGGUCUGAGCCUGGAGGAGU 1869 TTC TG-06-158 GGAGCAGGGCGCGUGAAGGG 1870 TTC TG-06-159 GCGCGCCCCUUCACGCGCCC 1871 TTC TG-06-160 CGCGCCCUGCUCCUGAACUU 1872 TTC TG-06-161 GCUCCUGCACAGUCCUCCCC 1873 TTC TG-06-167 CACUGAAUAGCGCAGCCGCA 1874 TTC TG-06-168 GUGGGAAGGUUCGCGGGGUU 1875 TTC TG-06-169 CGGGGUUGGGAGACCCGGAG 1876 TTC TG-06-170 UCGGCCUCCGGGUCUCCCAA 1877 TTC TG-06-171 CAGUACGUUCCAGGCAUUCA 1878 TTC TG-06-449 GCUGAAACAGAUGGAAUACU 1879 TTC TG-06-249 AAACCAAAUCGGAACCCACU 1880 TTC TG-06-386 UGUUGCCUGUAAUUGGAAUU 1881 TTC TG-06-122 CCUUUGUUUCUUCCCAGUAU 1883 TTC TG-06-127 UCCUCCUGCCUGGUACACAA 1884 TTC TG-06-137 GAAUGUACCUAUAUGACGUC 1885 TTC TG-06-431 CCCCGGCCUCCCAUCCCUAC 1886 TTC TG-06-433 CUUGGCACGAUCUUGGGGAC 1887 TTC TG-06-445 GAUUUGGUUUGGAAAACAUG 1888 TTC TG-06-446 CUCCAGGCCCUCCACCCUCC 1889 TTC TG-06-447 CACCCCGCCCCUGUCUCGGG 1890 TTC TG-06-355 CCAACCUCAGAAACUUGGAG 1891 TTC TG-06-356 GAGGUUGGAGAAGGUAGCCA 1892 TTC TG-06-359 AUCUACUCAUUCAGUCUAUG 1893 TTC TG-06-360 CCUCAUAGACUGAAUGAGUA 1894 TTC TG-06-361 GUCUAUGAGGGGAAGGCAAU 1895 TTC TG-06-362 AGCCAUUGCCUUCCCCUCAU 1896 TTC TG-06-363 UCCCUCUAAAAAUGCCACUU 1897 TTC TG-06-364 CUUCACCCCACAAGCAUCCU 1899 TTC TG-06-365 UGAGUUAUUCUUUCCCUUCA 1900 TTC TG-06-366 GGGCAGAGGGGACCAUCCUC 1901 TTC TG-06-367 UCCUUCAGGGCAGAGGGGAC 1902 TTC TG-06-368 CCUCCUGCCUUCCUCCUUCA 1903 TTC TG-06-369 GGGGCCUCCCCUCCACCACA 1904 TTC TG-06-370 GACUUACCCUGAUUGCUGGU 1905 TTC TG-06-371 CCCACACCCUGGCCAGCAAA 1906 TTC TG-06-372 CAAAUGGGUUCCCCCACACC 1907 TTC TG-06-373 CCCUCUUCUCAAAUGGGUUC 1908 TTC TG-06-374 UUUGGGCAACUUAUGGGAGG 1909 TTC TG-06-375 UCCCUCACCAAUUACCCCUC 1910 TTC TG-06-376 CAUCCCAUAUCCUCCACUGC 1911 TTC TG-06-377 AUCUGUCGCAGAGCCCAGGG 1912 TTC TG-06-378 GAGGGAGGUGGGGGAGGAAG 1913 TTC TG-06-379 UCCCCCACCUCCCUCAGAAC 1914 TTC TG-06-380 UGGGCCUGGGGACAUCCAUG 1915 TTC TG-06-381 UCAUUCUCUGGGUGCACGGU 1916 TTC TG-06-382 CUGGGUGCACGGUAACGACC 1917 TTC TG-06-383 CUGCUGCUCCCCUUUGGGAC 1918 TTC TG-06-384 UGCUCCAGGGGAGGCCUUUG 1919 TTC TG-06-385 UCAUCAAAGGCCUCCCCUGG 1920 TTC TG-06-450 UCGGCUGAAACAGAUGGAAU 1921 TTC TG-06-451 AUCUGUUUCAGCCGAAGAAA 1922 TTC TG-06-452 UUUCUUCGGCUGAAACAGAU 1923 TTC TG-06-453 GCCGAAGAAAAGAACCAGCU 1924 TTC TG-06-454 CCCCUGCCCCUUCAGCUGGU 1925 TTC TG-06-455 CGAGGCCCAUUGGCGUCCUU 1926 TTC TG-06-456 CUGCCUGAGUCCUAAAGGAC 1927 TTC TG-06-457 CACAGCCUGAGGGCCAGAAG 1928 TTC TG-06-458 GGCCCUCAGGCUGUGGGAAG 1929 TTC TG-06-459 UCCCGGGGCGAGACCACUAG 1930 TTC TG-06-460 CGGGGCGAGACCACUAGCUU 1931 TTC TG-06-461 AAGUAUUACCAGCCCAGGAC 1932 TTC TG-06-462 GUGUCCCCCAGCUUGGAGUC 1933 TTC TG-06-463 UCUCACUAGCUGUGGUGCUU 1934 TTC TG-06-464 CUCAUGGUCAACAGAACUUU 1937 TTC TG-06-465 GUUGACCAUGAGUGAACUUA 1938 TTC TG-06-466 CCUCUGCCCCAGGAGCUGUG 1939 TTC TG-06-467 GCCACUCACAGCUGCCUGCC 1940 TTC TG-06-468 GUCUGAGUGUGCUGGGUGGC 1941 TTC TG-06-469 UUGUUCCCUGAGCCUUUGAC 1942 TTC TG-06-470 CUGAGCCUUUGACUUUCUCG 1943 TTC TG-06-471 CGAGGGAUGUUGUGGGGUUG 1944 TTC TG-06-472 UAUCCUGGCCACAACCCCAC 1945 TTC TG-06-473 AGUUACCACUGCUCCAAAAC 1946 TTC TG-06-474 GGAAAUAGUGAGUACCCCAU 1947 TTC TG-06-475 GGUGGUCAUACAGCCUCUGC 1948 TTC TG-06-476 ACCAGAGAAGAAACAUCCUC 1949 TTC TG-06-477 AAACUUCCACCAGAGAAGAA 1950 TTC TG-06-478 UCUCUGGUGGAAGUUUGGAA 1951 TTC TG-06-479 CUGGUGGAAGUUUGGAACAG 1952 TTC TG-06-480 CACAAAAUAACCAUUGAAUG 1953 TTC TG-06-481 AUUCCCACAAAAUAACCAUU 1954 TTC TG-06-482 AAAUUCGAUUCCCACAAAAU 1955 TTC TG-06-483 UUCUAAAUUCGAUUCCCACA 1956 TTC TG-06-484 CAUGACUAAAAGAAAAGGAU 1957 TTC TG-06-485 UUUAGUCAUGAGAAACUGAG 1958 TTC TG-06-486 AAUGCACCUGGGGUGACCUC 1959 TTC TG-06-487 GGAAACCCAGUUCUAAUGCA 1960 TTC TG-06-488 AGAACUGACACUCCACUGCA 1961 TTC TG-06-489 AAGACCCAUCUGAAAAUGCC 1962 TTC TG-06-490 GAUGGGUCUUUGAAGCAUUA 1963 TTC TG-06-491 CAUGACACCAUCAUCGCUGA 1964 TTC TG-06-492 GCGAUGAUGGUGUCAUGAGA 1965 TTC TG-06-493 UGGUACCUCCUAAUCCUAGA 1966 TTC TG-06-494 AGGAUUAGGAGGUACCAUGA 1967 TTC TG-06-495 CAGCUCUGUAUCUUUGUUCA 1968 TTC TG-06-496 GACGGUGGAUAAAGAACUGU 1969 TTC TG-06-497 UUAUCCACCGUCUGAAAUGU 1970 TTC TG-06-498 CGUCAGGUGCUACCAUCAAA 1971 TTC TG-06-499 UCCAAGCAGACUCUGAUUGC 1972 TTC TG-06-500 AAGCAGACUCUGAUUGCCCA 1973 TTC TG-06-501 AAAAUCCAAACAAGUUCUGA 1974 TTC TG-06-502 ACUCUGACUGGAGUCCAAGA 1975 TTC TG-06-503 CUGGCUCCCCACAGCCUCAU 1976 TTC TG-06-504 UGAGGCUGUGGGGAGCCAGU 1977 TTC TG-06-505 UAAGCAGGUAUGUCUGCCUG 1978 TTC TG-06-506 CAGGGGUUCCUCCCCCAUGA 1979 TTC TG-06-507 AGGGAAAUCAUAGCGGGAGA 1980 TTC TG-06-508 CCCGCUAUGAUUUCCCUUGA 1981 TTC TG-06-509 CUUGAAGGCUGAUUAUUAAA 1982 TTC TG-06-510 CUUGCCAUGUGAUUGAUCUC 1983 TTC TG-06-511 GCCAUCAGUGAAAGCAGCUU 1984 TTC TG-06-512 UGGAGAGCUUGCAGAAUCAU 1985 TTC TG-06-513 GCUCAUGAUUCUGCAAGCUC 1986 TTC TG-06-514 UUUAUAUAGCAACACAAAAG 1987 TTC TG-06-515 UAAAGAGAGAGAAAAGUCAA 1988 TTC TG-06-516 CUCUCUUUAAGAAGUGUUGC 1989 TTC TG-06-517 GCCUGCAUUUGUAGAGCAUC 1990 TTC TG-06-518 CAGCAGGCCUAUGUCAUAGG 1991 TTC TG-06-519 GGUCCACGGUUUCCUGGCUC 1993 TTC TG-06-520 UGGCUCCAAAGCCCAUGAUU 1994 TTC TG-06-521 CUCAAUUUAUUCUGACUGGG 1995 TTC TG-06-522 GACUGGGGCAUGGGGGAGGG 1996 TTC TG-06-523 UCUGUACCUGCACCCAGCUC 1997 TTC TG-06-524 GGCAAAAAUGGGAACUACGG 1998 TTC TG-06-525 AAUGUGCCAUUCAGGCAAAA 1999 TTC TG-06-526 UUAUCAUCUACUAUACUCUG 2001 TTC TG-06-527 UCCAUGUCAUCAUGUUCCUC 2002 TTC TG-06-528 AUGUCAUCAUGUUCCUCCUU 2003 TTC TG-06-529 UCCUUGCAUGGGGCCAGGAU 2004 TTC TG-06-530 GCACCACCACGUAGGUGCCA 2005 TTC TG-06-531 AUGGCCUUCUUCCUGGCUUC 2006 TTC TG-06-532 CCAGGAAGCCAGGAAGAAGG 2007 TTC TG-06-533 UCCUGGCUUCCUGGUGAAGA 2008 TTC TG-06-534 UGGCUUCCUGGUGAAGAUGA 2009 TTC TG-06-535 UGGUGAAGAUGAGUGGCGAC 2010 TTC TG-06-536 CAAAAAGGGUGGCUCACCAG 2011 TTC TG-06-537 UGAUAGGGCUGGGCCACUGC 2012 TTC TG-06-538 GCAAUGGGCCUACUAAGCAC 2013 TTC TG-06-539 AUACAGAAAGCCAGUAGUUA 2014 TTC TG-06-540 GUAUAGAAUUCCCUUUAAGC 2015 TTC TG-06-541 CUUUAAGCCUGGCCAUGCCC 2016 TTC TG-06-542 AAUGAAGAUAGACGUACCAC 2017 TTC TG-06-543 GUCUCCUCGUCUUUCAAAUG 2018 TTC TG-06-544 UUUGAAAGACGAGGAGACUG 2019 TTC TG-06-545 GAGGGGACCACACAGACAGC 2020 TTC TG-06-546 UGUGCCAAUGCAUCUGCUGC 2021 TTC TG-06-547 GUAGGCAGCAGAUGCAUUGG 2022 TTC TG-06-548 UUUGAAGCUGUUGCUAUCCA 2023 TTC TG-06-549 CUCUGGAUAGCAACAGCUUC 2024 TTC TG-06-550 UUAGGACAGGGUGGCCCUCC 2025 TTC TG-06-551 GCCCCUCCACCGAAGAUGUG 2026 TTC TG-06-552 GCACCACCUUCUAGCCCACC 2029 TTC TG-06-553 AGCCCACCUCGUUUCCUGGC 2030 TTC TG-06-554 UGGCCUCUAACUUGAUGAGA 2031 TTC TG-06-555 CACUGAUUCUCCACAUGGCA 2032 TTC TG-06-556 CCACAUGGCAGGCGGUGCUU 2033 TTC TG-06-557 UAGCCUCCUGCAGACAGUGA 2034 TTC TG-06-558 GCCACGGUAUGCUGAGCUGA 2035 TTC TG-06-559 AAGGCAGGGACCCUGUCAAU 2036 TTC TG-06-560 AUCAGUCUGCUCAACACACG 2037 TTC TG-06-561 CCUCUGCAGCACACAGUAGG 2038 TTC TG-06-562 CUUGAGCUUACAGUUCAGGA 2039 TTC TG-06-563 GGAGGAGAGACUGACCAGUG 2040 TTC TG-06-564 UCUCUCCCCAAGGCUGAGCA 2041 TTC TG-06-565 UCGUCUCUCCCCAAGGCUGA 2042 TTC TG-06-566 GCUUGGCCUACUCUCCUCUG 2043 TTC TG-06-567 GUUUGCCUUCUGCUUGGCCU 2044 TTC TG-06-568 AACUGGGCAUCAUACCAGCC 2045 TTC TG-06-569 AGUCCUGCCCAGCCCCUUAU 2046 TTC TG-06-570 UCUAUCUGUAAACCAGUUAU 2047 TTC TG-06-571 UUUAAGGACUAAAUGAGGUC 2048 TTC TG-06-572 CGACCUCAUUUAGUCCUUAA 2049 TTC TG-06-573 GUACCUAGCAUCUGCUGAAU 2050 TTC TG-06-574 GCAGAUGCUAGGUACGGAAA 2051 TTC TG-06-575 GCGAGUUUCCGUACCUAGCA 2052 TTC TG-06-576 UAACCUGCCCCCACUUCAGC 2053 TTC TG-06-577 UAUCCCCAGAGGCUUCUUAA 2054 TTC TG-06-578 GACCUAAGUGGUAACAGCCU 2055 TTC TG-06-579 CUCUCCUUACCCUUCAGACC 2056 TTC TG-06-580 CUCUGCCCAGUUGGAGCCGG 2057 TTC TG-06-581 CCGCUGCCAUGCGCAGGGAG 2059 TTC TG-06-582 CCAGCCUGCUCAGUGCAAAC 2060 TTC TG-06-583 GUUUGUCCCCUAGUCUAUUC 2061 TTC TG-06-584 CUCCAUGUCCAUCCUGCCCA 2062 TTC TG-06-585 GAACUUGACAUCUUGUCUGG 2063 TTC TG-06-586 GAAUCCUUCCUAAAAGGAAU 2064 TTC TG-06-587 UAAAAGGAAUCCUCUAUGCC 2065 TTC TG-06-588 GCAGGCCUUGAAGUUGCCCC 2066 TTC TG-06-589 ACGGGAUGCUCUGGGCAAAG 2067 TTC TG-06-590 UCCGCCCGGUACCGUGGAGG 2068 TTC TG-06-591 GCUCAGAUGGGGUGGGGCAG 2069 TTC TG-06-592 UGUUGCCCCACAAAUGCAAA 2070 TTC TG-06-593 AAAUGGUGAGAAAAGGAAAA 2071 TTC TG-06-594 CACCAUUUGGAAUGGCCCAG 2072 TTC TG-06-595 AAGGUAAGUGCAGCCUGCUG 2073 TTC TG-06-596 UCUGAUGGUGACUCUACCUA 2074 TTC TG-06-597 UGGGGCUCCCUGCCUGGGCC 2075 TTC TG-06-598 CUCUAGGCCUUUCUUUCCAC 2076 TTC TG-06-599 UUCCACUGUUCCCUCUGCCU 2077 TTC TG-06-600 CUCUGCCUGGUGUGGCCUGG 2078 TTC TG-06-601 AUAGCCAGGCCACACCAGGC 2079 TTC TG-06-602 AUGGCCGCUCCUCCUCCCUC 2080 TTC TG-06-603 AGCAGGGACUUGCAGGUGGC 2081 TTC TG-06-604 CCGACUGAAUUGCCACCUGC 2082 TTC TG-06-605 GUCGGGGAAGACUCUAGAUG 2083 TTC TG-06-606 CUCCUCAGGCCAGGUGCAUC 2084 TTC TG-06-607 UUCUCUCCUCAGGCCAGGUG 2085 TTC TG-06-608 AGUUGGACUGUGUUAAGUUU 2086 TTC TG-06-609 CCCCAAGCUCUGCGUCCUGG 2087 TTC TG-06-610 CCCCUUACCGGUUCUCGUGG 2088 TTC TG-06-611 CAAGAUCCCACGAGAACCGG 2089 TTC TG-06-612 CGUGGGAUCUUGGGAAGCCA 2090 TTC TG-06-613 UCUAUGAGCUUUGUCGUUCU 2091 TTC TG-06-614 AUGAGCUUUGUCGUUCUUGU 2092 TTC TG-06-615 CACAGGUUGCUGUGAGACUC 2093 TTC TG-06-616 ACUCAUUGGAGUCUCACAGC 2094 TTC TG-06-617 AAAAGCAUCUGCACAUCCUU 2095 TTC TG-06-618 CUUCCAAAAGCAUCUGCACA 2096 TTC TG-06-619 CCACACCCAGCCCACCAGCU 2097 TTC TG-06-620 GCUGCUGCCACCCUUGCCAG 2098 TTC TG-06-621 UACAGAGGAGCUGUCCUCUC 2099 TTC TG-06-622 UUUCCAACCAGGUGAGACCU 2100 TTC TG-06-623 AACCAGGUGAGACCUCUUCA 2101 TTC TG-06-624 GUGGAGCCCUGGAGCCCCCU 2102 TTC TG-06-625 CAGUCCCUGCCUGGACUGGG 2103 TTC TG-06-626 AACCGAGGGGGUGGUAAUUU 2104 TTC TG-06-627 CUUGCCUCUUCCGUGUCCCA 2105 TTC TG-06-628 GUGUCCCAACAGCGGCAGCA 2106 TTC TG-06-629 UUGUCUCAUAAGGACACUUG 2108 TTC TG-06-630 GGCCCAAGGGAGGAGGGA 2109 TTC TG-06-631 CUCCUCUCCCUUGGGCCAGA 2110 TTC TG-06-632 UCCCCAUAGGAUGCCCAUGG 2111 TTC TG-06-633 CCAUAGGAUGCCCAUGGAUU 2112 TTC TG-06-634 CGCUUCCCCCGAGUGUCUGU 2113 TTC TG-06-635 CCCGAGUGUCUGUGGGAGGU 2114 TTC TG-06-636 ACACCCCUGCCUGCCAUUCC 2116 TTC TG-06-637 GGAGUCAUAUCAAGGGCUUG 2117 TTC TG-06-638 UCCAGCCAAGCCCUUGAUAU 2118 TTC TG-06-639 UCAUUCAUGGGGAGGCCACA 2119 TTC TG-06-640 UUCAUGGGGAGGCCACAAGC 2120 TTC TG-06-641 UGGGGAGGCCACAAGCAUGG 2121 TTC TG-06-642 CUGUCAGGUAUUGAGUGAGG 2122 TTC TG-06-643 UUCACAUUUGUAUCCCCAGC 2124 TTC TG-06-644 UUUCAUUCACAUUUGUAUCC 2125 TTC TG-06-645 GGAUACACUGUUGGGGGAAG 2126 TTC TG-06-646 CCCAACAGUGUAUCCAGAAG 2127 TTC TG-06-647 UUAAACAAAUGCUACUUGAC 2128 TTC TG-06-648 GAGCUCUCAGCCUAUCCAGA 2129 TTC TG-06-649 GGAUAGGCUGAGAGCUCUGA 2130 TTC TG-06-650 GCUGGAUACACAGGCUCGCA 2131 TTC TG-06-651 UGAUCCUUCUGCUGGAUACA 2132 TTC TG-06-652 UGGAGGAAGCGCUGUUCUAG 2133 TTC TG-06-653 AGCCAAGACCUACGGGGGCA 2134 TTC TG-06-654 ACUGCUUGGACACCGUCCCC 2135 TTC TG-06-655 UUCCACUGCUUGGACACCGU 2136 TTC TG-06-656 AUCCACGUUCAUUCCACUGC 2137 TTC TG-06-657 GCACAGGCCACACUCCCUCC 2138 TTC TG-06-658 CAUCUGACCUGGAGGCCACC 2139 TTC TG-06-659 CAGGCGUAUGGCAGGGCUGC 2141 TTC TG-06-660 CUCUCACCAGGCAGCCCUGC 2142 TTC TG-06-661 AGCCUCUGGAAAGCUCUGUG 2143 TTC TG-06-662 UGAGGCCACCUUGCCACGCU 2144 TTC TG-06-663 CUGUGACAGCGUGGCAAGGU 2145 TTC TG-06-664 AAACCCCCGGGUGAGGCAGG 2146 TTC TG-06-665 UAGGCCUCCCUCUAUCUUCC 2147 TTC TG-06-666 AGUGAGAGGCUGGAAAGGGG 2148 TTC TG-06-667 UUCCAGUGAGAGGCUGGAAA 2149 TTC TG-06-668 AGCCUCUCACUGGAAGGAAG 2150 TTC TG-06-669 UGCCCGCUCACACCAGCCCA 2151 TTC TG-06-670 AAGUGCAGGCGGUUCCUGCC 2152 TTC TG-06-671 AUCUGCCGCUGGGGCCACAC 2153 TTC TG-06-672 GCUGCAAGAUGAUGUGACCA 2154 TTC TG-06-673 GGUCCCACCCGUGCUUGGGA 2155 TTC TG-06-674 UCGUCGUGUGUGAGGAGGGA 2156 TTC TG-06-675 CUCCUCACACACGACGAGGA 2157 TTC TG-06-676 AGUCCAUGUUCCUCGUCGUG 2158 TTC TG-06-677 GCUCGAUGUGCGUUGUGUGG 2159 TTC TG-06-678 CUCCCUGGACGUAAGCCACA 2160 TTC TG-06-679 AGAUAAGCCUGUAUUCCCCG 2161 TTC TG-06-680 CCGGGCUGUCUGCCAAUGUA 2162 TTC TG-06-681 AGGCUGACGGGACAACUGGA 2163 TTC TG-06-682 CUCAGAGCUUCCAGGCUGAC 2164 TTC TG-06-683 UGAGCACCUGUAUCCCCUGC 2165 TTC TG-06-684 ACAUUCCAUAGCCCAGCUCA 2166 TTC TG-06-685 AUAGCCCAGCUCAAUAUCUA 2167 TTC TG-06-686 UCUAGGGUGGCGAGCACUGU 2168 TTC TG-06-687 GGUCGGAGCUCACAGCGCUC 2169 TTC TG-06-688 CAGUGUAGCACCGGGCACAU 2170 TTC TG-06-689 CUCAUCAAUAGGCAUCUACC 2171 TTC TG-06-690 GUAGCAUGCUGAGCAGGUCC 2172 TTC TG-06-691 UCUGUAGCAUGCUGAGCAGG 2173 TTC TG-06-692 UGCCGAGACUGGAGGCCUUA 2174 TTC TG-06-693 GACUGUGCCCAGACCUACUA 2175 TTC TG-06-694 GGCAAGGUCGGCCCCCAGUC 2176 TTC TG-06-695 UGUGGGAUCUCAGAGCAGCC 2177 TTC TG-06-696 GUGGCUGCUCUGAGAUCCCA 2178 TTC TG-06-697 CCAAAUAAGGUCUGGCUCAC 2179 TTC TG-06-698 CUCAUAAAGACACAUCACAG 2180 TTC TG-06-699 CCAAUCUCCACCUCUGUCUC 2181 TTC TG-06-700 UCCCAGAGCCUUCGCUGCUG 2182 TTC TG-06-701 CAGAGCCUUCGCUGCUGAGU 2183 TTC TG-06-702 CAGACUCAGCAGCGAAGGCU 2184 TTC TG-06-703 CUGCUGAGUCUGGGAAUUUG 2185 TTC TG-06-704 GUCACAAAUUCCCAGACUCA 2186 TTC TG-06-705 UAUGGCUUCGGUCACAAAUU 2187 TTC TG-06-706 CAUCUCAGGACAGGUGAGCA 2188 TTC TG-06-707 GAAGUGCCAUCUGAACAGCA 2189 TTC TG-06-708 GAUGGCACUUCAGAAGACUC 2190 TTC TG-06-709 GAAGACUCAGGAGACCCUGG 2191 TTC TG-06-710 UGGCUGCAGGGGUUCCAGGG 2192 TTC TG-06-711 GCUGGCGCAGGACUCUGCUA 2193 TTC TG-06-712 GGCAGGUUGCUGCUAGCAGA 2194 TTC TG-06-713 UUUGGCCUGGCUGAGAGUUU 2195 TTC TG-06-714 GAGACCUGCGCUGGAGCGGA 2196 TTC TG-06-715 UCUCUCCCUUCUCCAUCCAG 2197 TTC TG-06-716 CCAUCCAGCAGGCUGGACCU 2198 TTC TG-06-717 CCUAUACCCUAACCUUUGUC 2199 TTC TG-06-718 CCAAGAAGCAGGCACUGGGG 2200 TTC TG-06-719 CUGAGGUUGUGACUCGUGUG 2201 TTC TG-06-720 GCCUCACACGAGUCACAACC 2202 TTC TG-06-721 CCACUCCACAAGAUACCAGG 2203 TTC TG-06-722 UGUCUCUAAAAUGAGCCGGC 2204 TTC TG-06-723 CCUUUGAGUCUUUAAAUCUU 2205 TTC TG-06-724 CAGCUCACCGAGUCUGCAAG 2206 TTC TG-06-725 UGCAGACUCGGUGAGCUGUG 2207 TTC TG-06-726 UAUGAAGGAGCAGGAUGACU 2208 TTC TG-06-727 UGGCAGUAGCAUUGCCAGCU 2209 TTC TG-06-728 CCAGUGCCUGGGAUGUGCUC 2211 TTC TG-06-729 GUGUGUUAACUAUAAGGUUG 2212 TTC TG-06-730 AAGGCGACAUUUGUGGGAGA 2214 TTC TG-06-731 CGGUGGUCACUCUGUAUGCU 2215 TTC TG-06-732 CGAAGUCGGUGACCAUGACC 2216 TTC TG-06-733 AGAAUGUGCCCGAGGAGGAC 2217 TTC TG-06-734 ACAGACAGGUAAGCACGGCC 2218 TTC TG-06-735 CACAACUGAUGGCAGGGAGG 2219 TTC TG-06-736 UCCAUCCAGCCACCUGCUGA 2220 TTC TG-06-737 CAUGUGGUCCUUGUGUUCGU 2221 TTC TG-06-738 UCGAGCAGGCCAGCAAGUGU 2222 TTC TG-06-739 CUUGGCAGUUGAGCACGCGC 2223 TTC TG-06-740 UGGUUAGGAGACAUUAGCUC 2224 TTC TG-06-741 CUGGCUCUGCGGCAGAGGCU 2225 TTC TG-06-742 UCUGCACUCGUGGCCACUGG 2226 TTC TG-06-743 CAGUGGCCACGAGUGCAGAU 2227 TTC TG-06-744 UCAUCUGCACUCGUGGCCAC 2228 TTC TG-06-745 GCCUUGACCCUGGGUCAAUG 2229 TTC TG-06-746 ACAGUUCCACACUGCUCCCC 2230 TTC TG-06-747 AUGUGGAGGGACUCGAGGUG 2231 TTC TG-06-748 AUCUUCUGCUUGCAUACCUC 2232 TTC TG-06-749 GCUUGCAUACCUCUGAGACC 2233 TTC TG-06-750 AGACUGUGACUACAUUUAGU 2235 TTC TG-06-751 CAACUGGACUUCAUAGGGGG 2236 TTC TG-06-752 CUGUCCACCGGCCCCCAGAG 2237 TTC TG-06-753 UUCUGUGGCCAGACAUUGAG 2238 TTC TG-06-754 GUGGCCAGACAUUGAGCAAG 2239 TTC TG-06-755 UCAACUAUUUAGCAGCUACG 2240 TTC TG-06-756 CCAGCUUGAGCUGUGCGACC 2241 TTC TG-06-757 CAGCGAGUUCCCCAGCUUGA 2242 TTC TG-06-758 ACUGUAAGCCCCAGCCCCCA 2243 TTC TG-06-759 CAGCCUUUGAAACAGGCUAG 2244 TTC TG-06-760 AAGGCUGAGAAGGCCCAGGA 2245 TTC TG-06-761 GCCCUGAGACUUUCCUACAG 2246 TTC TG-06-762 UGCCUUUAAAACUGAACAUC 2247 TTC TG-06-763 GCCCCAUCAGGUGACCCCUU 2248 TTC TG-06-764 GGAACUGACCUGACUGAGCC 2249 TTC TG-06-765 UGAAGGUCAGUGUGUGGAGG 2250 TTC TG-06-766 GGGAAAGAACAGCCUCCCCU 2251 TTC TG-06-767 UUCCCAGAAAGGCCUUCCCC 2252 TTC TG-06-768 CCAGGCCUGGUGCUCUGGCC 2254 TTC TG-06-769 CCAGGAAGUCCUCCAGAGGC 2255 TTC TG-06-770 UGGAGAAGUCCCUUCUUUGG 2256 TTC TG-06-771 UUGGGGUCCCAGUCAGUGUA 2257 TTC TG-06-772 CAUACACUGACUGGGACCCC 2258 TTC TG-06-773 UCCUCUGUAAGCAGGUGAUU 2259 TTC TG-06-774 UCACCUCCACACUCAGUGCC 2260 TTC TG-06-775 CCUCCACACUCAGUGCCACU 2261 TTC TG-06-776 AGCUCCAUCCUUGGCCACGG 2262 TTC TG-06-777 GGAUCCUGGCCUCUAAGAUG 2263 TTC TG-06-778 CCCCUGUCAAAUGAGGUGUG 2264 TTC TG-06-779 AGGUCCCCUUGCACAUCAGG 2265 TTC TG-06-780 AACCCUCUGCCAGCCUGUGA 2266 TTC TG-06-781 GGCCUCAGUGGCACUCCACU 2267 TTC TG-06-782 UUCUGGCCUCAGUGGCACUC 2268 TTC TG-06-783 UUAGACAAUCCUGGGAGCAA 2269 TTC TG-06-784 AGUUUGUGUGGGUCCAACAC 2271 TTC TG-06-785 CCCGCCACUCUCUGAGGCCU 2272 TTC TG-06-786 CCACCCAACUUCCCCCGCCA 2273 TTC TG-06-787 CAUUCCUCUGAGGCUCAUCC 2274 TTC TG-06-788 UCUGAGGCUCAUCCUCUUGG 2275 TTC TG-06-789 AUGAAAGAGAGGCACCAAGA 2276 TTC TG-06-790 UGGAAAGGGAUAAUAAGGUU 2277 TTC TG-06-791 UAUAUUCAGACGCUUAGACA 2278 TTC TG-06-792 GACGCUUAGACAGAGCCAGG 2279 TTC TG-06-793 CUGUGCCUGGCUCUGUCUAA 2280 TTC TG-06-794 AGCUCAUGUGACCGUGCGGG 2281 TTC TG-06-795 GCAGGAAUUGCCAGCCUGCC 2283 TTC TG-06-796 UGCAGAAUUUGAUGUUGCAA 2284 TTC TG-06-797 UUCUUCCCUGGGAGGCCUCA 2286 TTC TG-06-798 UCCCUGGGAGGCCUCAGUCU 2287 TTC TG-06-799 CUGGGAGGCCUCAGUCUGUU 2288 TTC TG-06-800 CAGCAGCAUUUCCACUGGCU 2289 TTC TG-06-801 CAGCCCCUCCCUUACAGGCA 2290 TTC TG-06-802 UCCCAGGCCUGGAGUUUAUU 2291 TTC TG-06-803 GAAUAAACUCCAGGCCUGGG 2292 TTC TG-06-804 GAAAAGCCAGCUGGUCCAGC 2293 TTC TG-06-805 GGGACGAUGCCUGCCUCUAC 2294 TTC TG-06-806 UGUGGCACGUGGGCUUCUUG 2295 TTC TG-06-807 UGUGGCACGUUCCUGGAGGC 2296 TTC TG-06-808 GCCUCCAGGAACGUGCCACA 2297 TTC TG-06-809 UGGAGGCCGAACCCUUCUGG 2298 TTC TG-06-810 AAAGCCAGAAGGGUUCGGCC 2299 TTC TG-06-811 GGCUUUGGAAGGAGUCGUCA 2300 TTC TG-06-812 UCCCCAGCCUCCCGCAUGGC 2301 TTC TG-06-813 AGCCCCUUCCUCCCCAGCCU 2302 TTC TG-06-814 GACUCUGCGAUGAUGGAGGU 2303 TTC TG-06-815 GGAGGGAGAGUGGCCAGGAC 2304 TTC TG-06-816 GGUACCCGCCCCCUCCCCAU 2305 TTC TG-06-817 CAUUAGCCUUGCUCAAGUUG 2306 TTC TG-06-818 UCGGGUGAGAGGUCUGAGCC 2307 TTC TG-06-819 CCUAGAUACCCGCAGUGUCC 2308 TTC TG-06-820 UUCCCCUAGAUACCCGCAGU 2309 TTC TG-06-821 AGACUUGAAGUGGAACAAGA 2310 TTC TG-06-822 ACUUCAAGUCUGGAACUUCA 2311 TTC TG-06-823 AGUCUGGAACUUCAAGUCUG 2312 TTC TG-06-824 AGUCUGUGUGUGUGCGUGCG 2313 TTC TG-06-825 GCCGCCAGGGAAGCCUUGAU 2314 TTC TG-06-826 CUGGCGGCCGAAUUUAAAGG 2315 TTC TG-06-827 CCCCGUGGCACUUAGAACCA 2316 TTC TG-06-828 GUGUAUCAAGUGGUUCUAAG 2317 TTC TG-06-829 UAUCUCCCCCACUAGGAUGU 2318 TTC TG-06-830 CAAUGCCCCUGUGGAGUUUA 2319 TTC TG-06-831 UUAGGAGGGGACAUUUGAGU 2320 TTC TG-06-832 AGGUCAUCACAGUUGGGGCC 2322 TTC TG-06-833 UCUCUGGUCCCCCUGCACAG 2323 TTC TG-06-834 AGGACACUCAGUCUGAUGAG 2324 TTC TG-06-835 UCUGUGCACUCGCCUCCCUC 2325 TTC TG-06-836 CUGACCCUCGGGCUGGGCAG 2326 TTC TG-06-837 UCCAUCAAGCUCCCGAUCAA 2327 TTC TG-06-838 CCUGGCCGAUAAGACAGCAA 2328 TTC TG-06-839 CGAAGGUAAGCCGCCUGCAG 2329 TTC TG-06-840 AGAAGAGAGCUUAGUGUCUG 2330 TTC TG-06-841 CCCCUGCACCAGGCAUUGCA 2331 TTC TG-06-842 CUGCCAAAGAUGUCAUCAAU 2332 TTC TG-06-843 CUGAGGACCAGCGGGUACUG 2333 TTC TG-06-844 CCCUUGUCUGUGUAAGGAGG 2334 TTC TG-06-845 GAGCCCCAUUCUCAUUUAAU 2336 TTC TG-06-846 UUAAGCCCUCCUCUCUCCUA 2337 TTC TG-06-847 UGGUAGGAGAGAGGAGGGCU 2338 TTC TG-06-848 GUGGAGGUCCCCUCACUCCC 2339 TTC TG-06-849 CACUCCUGGAGAAACUGGAG 2340 TTC TG-06-850 CCAGGAGUGGGAAGCGGCGG 2341 TTC TG-06-851 GUAAGCUUACAGGGCUGGCC 2342 TTC TG-06-852 CGCGGCUGGGGGCUGCUGGA 2343 TTC TG-06-853 CGCUGUGGCUCUCUCCAGCA 2344 TTC TG-06-854 UAGAGGCUGCAGGACAGUGC 2345 TTC TG-06-855 AUGCUCACCUGGGUGUGAGC 2346 TTC TG-06-856 ACCAAAAUGCUGCAACCAGA 2348 TTC TG-06-857 GGCUUGAGGAGAUGGGGUCU 2349 TTC TG-06-858 AAAUGGAGAUAGAGAGAAAU 2350 TTC TG-06-859 AAACACAGAGUGGUUUCAAA 2351 TTC TG-06-860 UCCAAACACAGAGUGGUUUC 2352 TTC TG-06-861 GCCAGUGGGCAGCUCUGAAU 2353 TTC TG-06-862 GAGCUGCCCACUGGCAGAAC 2354 TTC TG-06-863 UUGGGCAGGAGUGAGCUCCU 2355 TTC TG-06-864 GGGGACAAGGAGCUCACUCC 2356 TTC TG-06-865 AAGCCCAUUCUAAAGCAGAU 2357 TTC TG-06-866 AAAGCAGAUUCCCAUUUCCG 2358 TTC TG-06-867 CAUUUCCGUCUUUGACUCUA 2359 TTC TG-06-868 GUCUUUGACUCUAAGGCCCA 2360 TTC TG-06-869 CCUUAUGCACCCACUGCCCG 2361 TTC TG-06-870 AUACUCUGGUUCUGCCACUU 2362 TTC TG-06-871 AUGCCCUUUGAGCCUGGGCA 2364 TTC TG-06-872 CCUCAUCUCAGCUUCCUCCU 2365 TTC TG-06-873 UCCUCCAUAAGAGGGAAAAA 2366 TTC TG-06-874 GCCCCAGCCGGGGGCCUCUC 2367 TTC TG-06-875 AACCGAGGUCUGCCCUCCUU 2368 TTC TG-06-876 CCCUGCCGCUCUAUAGCUGU 2369 TTC TG-06-877 UCAUACAGAGAUGUUAAGUA 2370 TTC TG-06-878 AGCUAUCGUUACUAUUGUUU 2371 TTC TG-06-879 UCACCCGUUGCUUUAAAAUC 2372 TTC TG-06-880 ACACAGGGUAGCCCGUCAAU 2373 TTC TG-06-881 UGCUACCUGCCUGGGCAUGU 2374 TTC TG-06-882 GGUUGGGUGGAGCAUGAGAG 2375 TTC TG-06-883 AGGCUGGAGUCCCACUGUCU 2377 TTC TG-06-884 CCUGGACGUGCUCAGACGAG 2378 TTC TG-06-885 CGUCUGAGCACGUCCAGGGG 2379 TTC TG-06-886 CAAACCCUCAGUGGUUCCCC 2380 TTC TG-06-887 GGGCACAGCAGGCUGGCAUG 2381 TTC TG-06-888 UGAACAUCCAUCCUACAUGC 2382 TTC TG-06-889 GGAAAGAUGAGCAUAGUCAG 2383 TTC TG-06-890 CUCCAAGCCAGGACCCCUGA 2384 TTC TG-06-891 GGGGUCCUGGCUUGGAGGGA 2385 TTC TG-06-892 AAAGGGGAGCGUGAUUAGAG 2386 TTC TG-06-893 AACUGACAUGUGCCUGUGAG 2387 TTC TG-06-894 AUGACUUCAAACUGACAUGU 2388 TTC TG-06-895 UGGAGUCAGAUUCCAUGACU 2389 TTC TG-06-896 CUUAGCUGCAAAAUGGUGCU 2390 TTC TG-06-897 CUAUGUCCUAGCUGCAAGGG 2391 TTC TG-06-898 GAGCACAUGCCAGGCUCCAU 2392 TTC TG-06-899 CACGUGUCAUCUUCACAGUG 2393 TTC TG-06-900 CAGUGUCCCUGUGAGUAGGU 2394 TTC TG-06-901 CUUUCCAUCUUACAAAUGAG 2395 TTC TG-06-902 UAGCCAAUGGGUGUGUCUGA 2397 TTC TG-06-903 GAGGUGGUUCAGACAGACCC 2398 TTC TG-06-904 GACAGACCCCGGCUUACCCU 2399 TTC TG-06-905 AGGGUAAGCCGGGGUCUGUC 2400 TTC TG-06-906 GCCUGCUGGCUGCAUAGGGA 2401 TTC TG-06-907 ACCCAGAGAUGGAGACAGUG 2402 TTC TG-06-908 CAGGGAAAACACCAUCUUUC 2403 TTC TG-06-909 CUGAGAAACUAAGGCUCAGA 2404 TTC TG-06-910 UUGAGUUGUUUCUAGGUUUC 2405 TTC TG-06-911 AGGUUUCCUAGCUCUUGCCU 2406 TTC TG-06-912 UAGCUCUUGCCUCAGACCUU 2407 TTC TG-06-913 CAUCUGAGCUGGCUUUCCUC 2408 TTC TG-06-331 UCUGCCCCAGGCUGCAGCUC 2409 TTC TG-06-915 UGCUGCCAUGCCCCAGGUCU 2410 TTC TG-06-916 AGACCUGGGGCAUGGCAGCA 2411 TTC TG-06-917 AUGCUCCUUGACUUUGCAUU 2412 TTC TG-06-332 CCUGCUCCUGAGGGGCCGGG 2413 TTC TG-06-919 GUGUCAGUUUGUGCCACCAC 2414 TTC TG-06-920 GCCAUUGUGUGGACAGCAUG 2415 TTC TG-06-921 ACUCCAAGUGGAGUGGGGUA 2416 TTC TG-06-922 UUAUGCCAUAUUUAUGCACA 2417 TTC TG-06-923 GCAUGCAGCUCAGGCACCCC 2418 TTC TG-06-924 UGCCUUGCUGGUCAUGCUAA 2419 TTC TG-06-925 AGGGCUUGGCACAUGCCUUG 2420 TTC TG-06-926 AGAAGGCCCGGCUGUCACAU 2421 TTC TG-06-927 AGAAACCUGAGUGUAUAACU 2422 TTC TG-06-928 UUUAAGAGUGGACUCCUUAG 2423 TTC TG-06-929 GGUCUGGAUGGUGCAUUCCU 2424 TTC TG-06-930 UCGAGAGCAAAAGACAGUCU 2425 TTC TG-06-931 AGGUGCUCUUCCUGGCACUG 2426 TTC TG-06-932 CCCUCCUGGAGCAUCUAUUU 2427 TTC TG-06-933 UCCACUAAAAUAGAUGCUCC 2428 TTC TG-06-934 GUACUCUAUAUUAAUGGUUU 2430 TTC TG-06-935 UCCACCUUGUCAGCGAGACC 2431 TTC TG-06-936 CUGCCUCAUGGCCUGUUUCG 2432 TTC TG-06-937 AGGCAAAGCAAAGGCCCCCA 2433 TTC TG-06-938 UGGUACUCCCUGCACAUCCC 2434 TTC TG-06-939 CACCUCUCUGGUUUCCUGGU 2435 TTC TG-06-940 GAUCCAGGCCAGACCACACU 2436 TTC TG-06-941 CAGCUCAGCAGCUAGGCCAC 2437 TTC TG-06-942 GCCCAAGGUCCUUGUCAUCU 2438 TTC TG-06-943 GAGGGAUGAGUUUGGCACUG 2440 TTC TG-06-944 UGCCUGGUUUGAGGGCCGUG 2441 TTC TG-06-945 GCACCAUGGCUGACCAAACA 2442 TTC TG-06-946 GUGAGGCCAAAGUGCAGACG 2443 TTC TG-06-947 CCCUCCGACAUCCUUCUGUG 2444 TTC TG-06-948 UUUCUCCCUCCGACAUCCUU 2445 TTC TG-06-949 UUUCCUCGGGCUCUGGCAGG 2446 TTC TG-06-950 UCGGGCUCUGGCAGGUGACC 2447 TTC TG-06-951 CUGGUGCUGCCUGUAGUGCU 2448 TTC TG-06-952 GACUUGUCCCUCUCUCAGCC 2449 TTC TG-06-953 GCCUUUCCGGGGCUGCUGGC 2450 TTC TG-06-333 GGGGCUGCUGGCCUGGCCCU 2451 TTC TG-06-955 AGGCAAGGAGGCUGCCCCAC 2452 TTC TG-06-956 UGGCACCUCCACCUGGGGAG 2453 TTC TG-06-957 CCAUUCAAACAGGUCGAGCU 2454 TTC TG-06-958 AACAGGUCGAGCUGUGCUCG 2455 TTC TG-06-959 GCCCACGGACAUCGGCACAU 2456 TTC TG-06-960 GUGCCAGGCAUUCAAUCCUC 2457 TTC TG-06-961 AUCCUCAGGUCUCCACCAAG 2458 TTC TG-06-962 UCCCAUGGAUAGGGGAGGGG 2459 TTC TG-06-963 CAUGGAUAGGGGAGGGGGCG 2460 TTC TG-06-964 CCACAGUUAGCUGGAGAUGA 2461 TTC TG-06-965 GGAUGGCAUCUAGCCAGAGG 2462 TTC TG-06-966 UGAGCCACCUUUACUCUGCU 2463 TTC TG-06-967 UCCUCUGUGAAGUAGGGGUG 2465 TTC TG-06-968 CAGAGGAAGAAACCUGGAAC 2466 TTC TG-06-335 UGCUGUGUGAGCUUGGCAGG 2467 TTC TG-06-970 GGCUCAGUUCCUGCUGUGUG 2468 TTC TG-06-971 GAGCCAGCCCAAUCUGCGUU 2469 TTC TG-06-972 UACUUCACCCGGCUGGGCUC 2470 TTC TG-06-973 CCCGGCUGGGCUCCUCAUUU 2471 TTC TG-06-974 CAGCCUCACUGUUACCCGUA 2472 TTC TG-06-336 CCUUCCCAGCCUCACUGUUA 2473 TTC TG-06-976 UGGUCUGUGUUCCCCUUCCC 2474 TTC TG-06-977 GCCAUCACUCACCGAGCUUC 2475 TTC TG-06-978 AUGCCUGCAGGCAUCGUUCU 2476 TTC TG-06-337 GUUAUCACCCAGGCCUGAUU 2477 TTC TG-06-338 CUGGCCUGGCGGAGAUGCUU 2478 TTC TG-06-339 AAGGCAUGGUCGGGGGAGAG 2479 TTC TG-06-341 AGUUACAAAAGCAAAACAGG 2480 TTC TG-06-983 UAGUAUAAGCAAGGCAUCAC 2481 TTC TG-06-984 ACAAACUUACCCAGUUCCUA 2482 TTC TG-06-985 CAUCACCAGUACUUGCCCAA 2483 TTC TG-06-986 UAGGAUAGGCACAAGGAGUA 2484 TTC TG-06-987 UUCCUAGGAUAGGCACAAGG 2485 TTC TG-06-988 UGCAGCUCCCCUGACCACCC 2486 TTC TG-06-989 UUUCUCCCCAGUACCUGGCA 2487 TTC TG-06-990 CCCCAGUACCUGGCACACAG 2488 TTC TG-06-991 GUAAAUACUCGGGCCAGGCU 2489 TTC TG-06-992 GUAAUGAGCCAGUGCUAAGU 2490 TTC TG-06-993 UGAUUUAAAAUUCAGAACAG 2491 TTC TG-06-994 GAACAGUCUCAGAUUGGGUG 2492 TTC TG-06-995 UAAACACAAGCAAUCCUCCU 2493 TTC TG-06-996 AGACCCUGGGCAACAGUGAG 2494 TTC TG-06-997 AUGUGGGACCUCCAGGCCCU 2495 TTC TG-06-998 CCAGCCUUAGAGUGGAGGGC 2496 TTC TG-06-999 CUCUCUCUCUAGACAGCCCA 2497 TTC TG-06-1000 UUAUGACAUGGGCUGUCUAG 2498 TTC TG-06-1001 GACAAUUCAGUGUCAUAUAU 2499 TTC TG-06-1002 AUAUAUGACACUGAAUUGUC 2500 TTC TG-06-1003 GUGUCAUAUAUGGAAAGCAU 2501 TTC TG-06-1004 AGAGUUGACAGAUGGCAAUG 2502 TTC TG-06-1005 AGCCAUUGCCAUCUGUCAAC 2503 TTC TG-06-1006 AACGGGCAGAGAUAACACAG 2504 TTC TG-06-1007 GUGGAGGGGAAGGAGAGAGA 2505 TTC TG-06-1008 CCUCCACAGAAUCAAGUAUC 2506 TTC TG-06-1009 AACAUCACAGAGCUGUCAGG 2507 TTC TG-06-1010 UGCAGAGGGUUAGCAGGUGG 2508 TTC TG-06-1011 UCCCCGAUCCCUGGUGACCA 2509 TTC TG-06-1012 CUAUUUGCUAGUCCAGAUUU 2511 TTC TG-06-1013 UAGUGGCCAGCGUUGCUCAC 2512 TTC TG-06-1014 UGCUAGAUCCCACAGGUCUC 2513 TTC TG-06-1015 GCAUCCUCCUCUAAGCUUUG 2514 TTC TG-06-1016 UCUGCAAAGCUUAGAGGAGG 2515 TTC TG-06-1017 GGAUUCAUCUGCAAAGCUUA 2516 TTC TG-06-1018 CUGGAGGGGCUGACCAUUUC 2517 TTC TG-06-1019 UUUCUCUGGAGGGGCUGACC 2518 TTC TG-06-1020 UCUUAGUACUUUACACUAAU 2519 TTC TG-06-1021 CCCGCCCACUACGGCUGUGU 2520 TTC TG-06-1022 UAUAUCAGAACACAGCCGUA 2521 TTC TG-06-1023 UCAUAUAUCAGAACACAGCC 2522 TTC TG-06-1024 GAUAUAUGAAGAAUGGCGGC 2523 TTC TG-06-1025 AAAACAGAGUUGGAGUCUUG 2524 TTC TG-06-1026 UAGCUUCUAAGAGUAUAAAA 2525 TTC TG-06-1027 UGUGUGGCAAAGUUCUUAGC 2526 TTC TG-06-1028 AAAUUUGGUUCAUGUGUGGC 2527 TTC TG-06-1029 UCCCUAGUGUAUCUACUUCC 2528 TTC TG-06-1030 CUAGUGUAUCUACUUCCUUU 2529 TTC TG-06-1031 AAAGGAAGUAGAUACACUAG 2530 TTC TG-06-1032 UCUGCUACCUCUUGUGUAUA 2531 TTC TG-06-1033 AGUCUGCUUGUUCCAAGCUC 2532 TTC TG-06-1034 GAGCUUGGAACAAGCAGACU 2533 TTC TG-06-1035 AAGCUCAGAAGGUAACUGCA 2534 TTC TG-06-1036 CUGCUAACAUCGAGGUGUGU 2535 TTC TG-06-1037 UAAAACUGACUGUAGUUGUC 2536 TTC TG-06-1038 UUCCCAGGAUGCCUUCAACG 2537 TTC TG-06-1039 GCCCGUUGAAGGCAUCCUGG 2538 TTC TG-06-1040 CAGGAUGCCUUCAACGGGCA 2539 TTC TG-06-1041 ACGGGCAGAAUCGGCCAUCU 2540 TTC TG-06-1042 CAAGAAGCUCCCUUAGUGAG 2541 TTC TG-06-1043 UGAGAACACACAGCAUUAUU 2542 TTC TG-06-1044 UGCUCUGUUUGCAGACCAUU 2543 TTC TG-06-1045 AAAUGGUCUGCAAACAGAGC 2544 TTC TG-06-126 CCUUUUCAUCCUCCUGCCUG 2672 TTC TG-06-129 UUUUACACACCAUGUUCAAG 2675 TTC TG-06-148 GCCGGGCCCACCUUUUCAGU 2694 TTC TG-06-1046 UCUCUUACAUGGGGGGAAAC 2714 TTC c.gRNA modification

In another aspect, the present invention relates to gRNAs (sometimes referred to herein as gRNA variants) comprising modifications relative to a reference gRNA from which the gRNA is derived. The gRNA can be used in the long-term gene suppressor fusion protein system described herein. In some embodiments, the gRNA variants used in the disclosed systems comprise one or more nucleotide substitutions, insertions, deletions, or exchanges or replacement domains relative to the gRNA sequence disclosed herein, which improve the characteristics. Exemplary regions of modifications and exchange regions or domains include RNA triple helices, pseudoknots, scaffold stem loops, and extended stem loops. In some embodiments, the gRNA variants disclosed herein comprise at least one first exchange region from a different gRNA, resulting in a chimeric gRNA. A representative example of such a chimeric gRNA is guide 316 (SEQ ID NO: 1746), in which the extended stem loop of gRNA scaffold 235 (SEQ ID NO: 1745) is replaced by the extended stem loop of gRNA scaffold 174 (SEQ ID NO: 1744), wherein the resulting 316 variant is still able to form RNPs with long-term suppressor fusion proteins and exhibits improved properties compared to the parental 235 when assessed under comparable conditions in in vitro or in vivo assays.

All gRNAs that have one or more improved functions, features, or add one or more new functions when the gRNA scaffold variants are compared to the gRNA scaffold from which they are derived, while maintaining the functional properties of being able to complex with a long-term suppressor fusion protein and direct the ribonucleoprotein into a complex of an RNP and a PCSK9 target nucleic acid are contemplated to be within the scope of the present invention. In some embodiments, the gRNA has an improved feature selected from the group consisting of increased pseudostem stability, increased triple helical region stability, increased scaffold stem stability, extended stem stability, reduced off-target fold intermediates, increased binding affinity to a long-term suppressor fusion protein, and increased inhibitory activity when complexed with a long-term suppressor fusion protein, or any combination thereof. In some cases, the improved characteristics are assessed in an in vitro assay (including assays in each example). In other cases, the improved characteristics are assessed in vivo.

Table 10 provides exemplary gRNA variant scaffold sequences of the present disclosure, which are used as gRNA scaffolds or for generating gRNAs for use in the LTRP:gRNA systems of the present disclosure. In some embodiments, the gRNA variant scaffolds used in the system include a sequence selected from the group consisting of SEQ ID NOs: 1744 to 1746 or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto, wherein the gRNA variant is still capable of forming RNPs with the long-term suppressor fusion protein of the present disclosure. In some embodiments, the gRNA variant scaffolds used in the system include a sequence selected from the group consisting of SEQ ID NOs: 1744 to 1746. In some embodiments, the gRNA variant scaffold for the system comprises SEQ ID NO: 1744. In some embodiments, the gRNA variant scaffold for the system comprises SEQ ID NO: 1745. In some embodiments, the gRNA variant scaffold for the system comprises SEQ ID NO: 1746. It should be understood that in those embodiments in which the vector comprises a DNA encoding sequence of a gRNA, a thymine (T) base can replace the uracil (U) base of any of the gRNA sequence embodiments described herein. Similarly, any RNA sequence disclosed herein can be encoded by a DNA in which the uracil base is replaced by a thymine. In some embodiments, the present disclosure provides the chemically modified gRNA variants of Table 10 described below. In some embodiments, the gRNA comprises a scaffold comprising a sequence of SEQ ID NO: 1744 to 1746 and is chemically modified. Table 10 : gRNA scaffold sequence SEQ ID NO: Stent variant ID Nucleotide sequence 1744 174 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 1745 235 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCCGUAAGAGGCAUCAGAG 1746 316 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG

Additional gRNA variants contemplated for use in the systems of the present disclosure are selected from the group consisting of SEQ ID NOs: 1747 to 1821. In some embodiments, the gRNA comprises a scaffold comprising a sequence of SEQ ID NOs: 1747 to 1821 and is chemically modified.

The guide scaffold can be made by several methods including recombinant methods or solid phase RNA synthesis. However, when solid phase RNA synthesis is used, the length of the scaffold affects manufacturability, with longer lengths resulting in increased manufacturing costs, reduced purity and yield, and higher synthesis failure rates. For use in particle formulations, such as lipid nanoparticle (LNP) formulations, solid phase RNA synthesis of the scaffold is preferred in order to produce the quantities required for commercial development. Although previous experiments have identified gRNA scaffold 235 (SEQ ID NO: 1745) as having enhanced properties relative to gRNA scaffold 174 (SEQ ID NO: 1744), its increased length (in nucleotides) makes its use in LNP formulations less ideal due to synthetic manufacturing limitations. Therefore, alternative sequences are sought. In some embodiments, the disclosure provides gRNA variant scaffolds having improved manufacturability compared to the gRNA scaffolds from which they are derived. In some embodiments, the disclosure provides gRNAs wherein the gRNA scaffold and the linked targeting sequence have a sequence of less than about 115 nucleotides, less than about 110 nucleotides, or less than about 100 nucleotides. In a particular embodiment, the 316 gRNA scaffold (SEQ ID NO: 1746) has a shorter sequence than the 235 scaffold from which it is derived. The 316 gRNA scaffold was designed in which the scaffold 235 sequence was modified by domain swapping, in which the extended stem loop of scaffold 174 replaced the extended stem loop of scaffold 235, resulting in a chimeric gRNA scaffold 316 having the sequence ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG (SEQ ID NO: 1746), which has 89 nucleotides compared to the 99 nucleotides of gRNA scaffold 235. The resulting 316 scaffold has the additional advantage that the extended stem loop does not contain CpG motifs; a property that confers an enhanced reduced potential to elicit an immune response. In some embodiments, the shorter sequence length of the 316 scaffold confers a higher fidelity improvement in the ability to synthetically generate guides with the correct and complete sequence, as well as an enhanced ability to be successfully incorporated into LNPs. In some embodiments, the disclosure provides chemically modified gRNA 316 variants described below. d. Chemically modified gRNA

In some embodiments, the gRNA has one or more chemical modifications. In some embodiments, the chemical modification is the addition of a 2'O-methyl group to one or more nucleotides of the sequence. In some embodiments, the chemical modification is the replacement of a phosphorothioate bond between two or more nucleotides of the sequence. In some embodiments, the chemical modification is the replacement of a phosphorothioate bond between two or more nucleotides at each end of the gRNA. In some embodiments, the gRNA comprises the replacement of a phosphorothioate bond between two or more nucleotides at 1, 2, 3, or 4 nucleotides from the 5' end, 3' end, or both ends of the gRNA. In some embodiments, the gRNA comprises the addition of a 2'O-methyl group to one or more nucleotides of the gRNA. In some embodiments, one or more nucleotides located 1, 2, 3, or 4 nucleotides from the 5' end, 3' end, or both ends of the gRNA are modified by adding a 2'O-methyl group. In some embodiments, 1, 2, or 3 nucleotides before the 5' end of the scaffold (i.e., A, C, and U in the case of gRNAs 174, 235, and 316) are modified by adding a 2'O-methyl group and each of the modified nucleotides is linked to the adjacent nucleotide by a phosphorothioate bond. Similarly, the last 1, 2, or 3 nucleotides at the 3' end of the targeting sequence linked to the 3' end of the scaffold are similarly modified. In some embodiments, the present disclosure provides gRNAs with chemical modifications selected from the group consisting of SEQ ID NOs: 2948 to 2956, 2958 to 2966, and 2968 to 2976 as described in Table 22, or sequences having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In any of the foregoing, the 20 nucleotides on the 3' end of the sequence are replaced with a targeting sequence complementary to the PCSK9 target nucleic acid sequence, optionally selected from the group consisting of SEQ ID NOs: 1824 to 1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694 and 2714, but including chemical modifications on the 3' end of the gRNA. In some embodiments, the 20 nucleotides at the 3' end of the sequence are replaced with a targeting sequence complementary to a PCSK9 target nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1834, 1849, 1853, 1855 to 1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875, but including a chemical modification at the 3' end of the gRNA. In some embodiments, the 20 nucleotides at the 3' end of the sequence are replaced with a targeting sequence complementary to a PCSK9 target nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1855, 1867, and 1869, but including a chemical modification at the 3' end of the gRNA. In some embodiments, the gRNA with a chemical modification comprises a sequence selected from the group consisting of SEQ ID NOs: 2948 to 2956, 2958 to 2966, and 2968 to 2976, wherein 20 nucleotides on the 3' end are replaced with a targeting sequence selected from the group consisting of SEQ ID NOs: 1824 to 1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714, but with a chemical modification on the 3' end. In some embodiments, the chemically modified gRNA comprises a sequence of SEQ ID NO: 2968, wherein 20 nucleotides on the 3' end are replaced by a targeting sequence selected from the group consisting of SEQ ID NOs: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714, but with a chemical modification on the 3' end. In some embodiments, the chemically modified gRNA comprises a sequence of SEQ ID NO: 2968, wherein 20 nucleotides on the 3' end are replaced by a targeting sequence selected from the group consisting of SEQ ID NOs: 1855, 1867, and 1869, but with a chemical modification on the 3' end. In some embodiments, the modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 22780 to 22803. In some embodiments, the modified gRNA comprises a sequence selected from the group consisting of SEQ ID NO: 22788 to 22790. Schematic diagrams of the structures of gRNA variants 174, 235, and 316 are shown in Figures 7 to 9, respectively, and chemically modified versions of gRNA variants 235 and 316 are shown in Figures 10 to 14. In some embodiments, the gRNA with chemical modification exhibits improved stability compared to the gRNA without chemical modification. e. Formation of complex with suppressor fusion protein

After delivery or expression of the components of the system in the target cell, the gRNA variants are able to complex with the long-term suppressor fusion protein into RNPs and bind to the target nucleic acid of the PCSK9 gene. In some embodiments, the gRNA variants have an improved ability to form RNP complexes with the long-term suppressor fusion protein when compared to the reference gRNA. In some embodiments, the improved ribonucleoprotein complex formation can increase the efficiency of assembling functional RNPs. In some embodiments, greater than 90%, greater than 93%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% of the RNPs comprising the gRNA variants and their targeting sequences and the long-term suppressor fusion proteins disclosed herein are competent for gene inhibition of the target nucleic acid. VII. Polynucleotides and Vectors

The present invention provides polynucleotides encoding long-term suppressor fusion proteins and/or gRNAs that are useful in the inhibition and epigenetic modification of the PCSK9 gene. The present disclosure provides polynucleotides encoding mRNAs, such as DNA polynucleotides encoding corresponding mRNAs, that encode long-term suppressor fusion proteins.

The long-term suppressor fusion proteins disclosed herein or mRNA encoding long-term suppressor fusion proteins can be prepared by in vitro synthesis using conventional methods known in the art. Various commercial synthesis apparatuses are available, such as automated synthesizers from Applied Biosystems, Inc., Beckman, and the like. Naturally occurring amino acids or nucleotides (where appropriate) can be replaced by non-natural amino acids or nucleotides using a synthesizer. The specific order and manner of preparation will be determined by convenience, economic factors, desired purity, and the like. gRNA can also be produced synthetically; for example, by using the T7 RNA polymerase system known in the art.

Long-term suppressor fusion proteins and/or gRNAs can also be prepared by recombinantly generating a polynucleotide sequence encoding a long-term suppressor fusion protein or gRNA of any of the embodiments described herein using standard recombination known in the art, and incorporating the encoding gene into an expression vector suitable for a host cell. To produce an encoded long-term suppressor fusion protein and/or gRNA of any of the embodiments described herein, the methods include transforming a suitable host cell with an expression vector comprising the encoding polynucleotide, and culturing the host cell under conditions that cause or allow the resulting long-term suppressor fusion protein or gRNA of any of the embodiments described herein to be expressed or transcribed in the transformed host cell, and recovering by the methods described herein or by standard purification methods known in the art or as described in the examples. The polynucleotides and expression vectors of the present disclosure are made using standard recombinant techniques in molecular biology.

The long-term suppressor fusion proteins and/or gRNA disclosed herein can also be isolated and purified according to known recombinant synthesis methods. Lysates expressing the host can be prepared and purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography or other purification techniques. In most cases, the composition used will account for 50% or more by weight of the desired product, more usually 75% or more by weight, preferably 95% or more by weight, and for therapeutic purposes, usually 99.5% or more by weight, relative to contaminants involved in the preparation of the product and its purification method. Typically, the percentage will be based on total protein. Thus, in some cases, the long-term suppressor fusion protein or gRNA disclosed herein is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants or other macromolecules, etc.).

In addition, the present disclosure provides vectors comprising polynucleotides encoding the long-term suppressor fusion proteins and gRNAs described herein. In some cases, when the system is delivered in the form of suppressor fusion proteins and gRNAs or RNPs, the vector is used to express and recover the CasX and gRNA components of the LTRP:gRNA system. In other cases, as described more fully below, the vector is used to deliver the encoding polynucleotides to the target cell to inhibit and/or epigenetically modify the target nucleic acid. In some embodiments, the sequences encoding the long-term suppressor fusion proteins and gRNAs are templated on the same vector. In some embodiments, the sequences encoding the long-term suppressor fusion proteins and gRNAs are templated on different vectors. Suitable vectors are described, for example, in WO2023235818A2, WO2022120095A1, and WO2020247882A1, each of which is incorporated herein by reference. As described in WO2023235818A2, WO2022120095A1, and WO2020247882A1, any of a variety of suitable transcriptional and translational control elements may be used in the expression vector, depending on the host/vector system used, including perturbative and inducible promoters, transcriptional enhancer elements, transcriptional terminators, and the like.

The present disclosure provides polynucleotide sequences encoding the long-term suppressor fusion protein of any embodiment described herein. In some embodiments, the present disclosure provides DNA sequences encoding the long-term suppressor fusion protein for use in vectors. In some embodiments, the present disclosure provides mRNA sequences encoding the long-term suppressor fusion protein of any embodiment described herein, which are used in particle systems for delivery to cells. In some embodiments, the present disclosure provides an mRNA sequence encoding the long-term suppressor fusion protein of any embodiment described herein, which is used in LNP particle formulations for delivery to cells. In a specific embodiment, the present disclosure provides gRNA and mRNA sequences encoding the long-term suppressor fusion protein of any embodiment described herein, which are used in LNP particle formulations for delivery to cells. In some embodiments, the disclosure provides isolated polynucleotide sequences encoding a gRNA variant of any of the embodiments described herein, the RNA variant having a linked targeting sequence that is complementary to a PCSK9 target nucleic acid sequence.

In some embodiments, the disclosure relates to methods of generating polynucleotide sequences encoding long-term suppressor fusion proteins or gRNAs (including variants thereof) of any of the embodiments described herein, and methods of expressing proteins or RNAs transcribed from the polynucleotide sequences. In general, the methods include generating polynucleotide sequences encoding long-term suppressor fusion proteins or gRNAs of any of the embodiments described herein and incorporating the encoding genes into expression vectors. In some embodiments, the vectors are designed for transducing cells to inhibit and/or epigenetically modify PCSK9 target nucleic acids. Such vectors may include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors, plasmids, microcircles, nanoplasmids, DNA vectors, and RNA vectors. In other embodiments, the expression vector is designed for producing a long-term suppressor fusion protein, mRNA encoding the long-term suppressor fusion protein, or gRNA in a cell-free system or host cell. To produce the encoded long-term suppressor fusion protein or gRNA of any embodiment described herein in a host cell, the methods include transforming a suitable host cell with an expression vector comprising an encoding polynucleotide, and culturing the host cell under conditions that allow or permit the resulting long-term suppressor fusion protein or gRNA of any embodiment described herein to be expressed or transcribed in the transformed host cell, thereby producing the long-term suppressor fusion protein or gRNA, which is recovered by the methods described herein (e.g., the methods described in the examples below) or by standard purification methods known in the art. The polynucleotides and expression vectors of the present disclosure are made using standard recombinant techniques in molecular biology.

According to the present disclosure, a nucleic acid sequence encoding a long-term suppressor fusion protein or gRNA of any of the embodiments described herein is used to generate a recombinant DNA molecule directed for expression in an appropriate host cell. Several cloning strategies are suitable for carrying out the present disclosure, many of which are used to generate a construct comprising a gene encoding a long-term suppressor fusion protein or gRNA disclosed herein or a complementary sequence thereof. In some embodiments, a cloning strategy is used to generate a gene encoding a construct comprising nucleotides encoding a long-term suppressor fusion protein or gRNA. In some embodiments, a gene is used (e.g., as part of a vector) to transform a host cell to express the gene, e.g., to express a long-term suppressor fusion protein or gRNA.

In one method, a construct is first prepared that contains a DNA sequence encoding a long-term suppressor fusion protein or gRNA. Exemplary methods for preparing such constructs are described in the Examples. The construct is then used to generate an expression vector suitable for transforming a host cell (such as a prokaryotic or eukaryotic host cell) to express and recover the protein construct in the presence of a long-term suppressor fusion protein or gRNA. When necessary, the host cell is E. coli. In other embodiments, the host cell is a eukaryotic cell. Eukaryotic host cells can be selected from baby hamster kidney fibroblasts (BHK) cells, human embryonic kidney 293 (HEK293) cells, human embryonic kidney 293T (HEK293T) cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, fusion tumor cells, NIH3T3 cells, CV-1 (simian) SV40 genetic material source (COS) cells, HeLa cells (HeLa), Chinese hamster ovary (CHO) cells, yeast cells, or other eukaryotic cells known in the art to be suitable for producing recombinant products. Exemplary methods for producing expression vectors, transforming host cells, and expressing and recovering long-term suppressor fusion proteins or gRNAs are described in the Examples.

Genes encoding long-term suppressor fusion proteins or gRNA constructs can be prepared in one or more steps, either completely synthetically or by a combination of synthetic methods and enzymatic methods such as restriction enzyme-mediated cloning, PCR, and overlapping extension, including methods more fully described in the Examples. The methods disclosed herein can be used, for example, to join sequences of polynucleotides encoding various components into genes of desired sequences. Genes encoding polypeptide compositions are assembled from oligonucleotides using standard techniques for gene synthesis.

In some embodiments, the nucleotide sequence encoding the long-term suppressor fusion protein is codon optimized. This type of optimization may require mutations in the coding nucleotide sequence to mimic the codon preference of the intended host organism or cell while encoding the same protein. Thus, the codons may change, but the encoded protein remains unchanged. For example, if the intended target cell for the long-term suppressor fusion protein is a human cell, a human codon-optimized nucleotide sequence encoding the long-term suppressor fusion protein may be used. As another non-limiting example, if the intended host cell is a mouse cell, a mouse codon-optimized nucleotide sequence encoding the long-term suppressor fusion protein may be generated. Genetic design may be performed using an algorithm that optimizes codon usage and amino acid composition of a host cell suitable for producing a long-term suppressor fusion protein or gRNA. In one disclosed method, a polynucleotide library encoding components of the construct is generated and then assembled as described above. The resulting genes are then assembled and used to transform host cells and produce and recover long-term suppressor fusion proteins or gRNA compositions to assess their properties or to modify the PCSK9 target nucleic acids described herein.

In some embodiments, the nucleotide sequence encoding the gRNA is operably linked to a control element, such as a transcriptional control element, such as a promoter. In some embodiments, the nucleotide sequence encoding the long-term suppressor fusion protein is operably linked to a control element, such as a transcriptional control element, such as a promoter. In some cases, the promoter is a continuously active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an induced promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (e.g., a promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcriptional control element can be functional in eukaryotic cells, such as hepatocytes or hepatic sinus endothelial cells.

Non-limiting examples of Pol II promoters that are operably linked to a polynucleotide encoding a long-term suppressor fusion protein disclosed herein include, but are not limited to: EF-1α, EF-1α core promoter, Jens Tornoe (JeT), cytomegalovirus (CMV) promoter, CMV immediate early (CMVIE) promoter, CMV enhancer, herpes simplex virus (HSV) thymidine kinase, early and late simian virus 40 (SV40), SV40 enhancer, long terminal repeats (LTR) from retrovirus, mouse metallothionein-I, adenovirus major late promoter (Ad MLP), CMV promoter full-length promoter, minimal CMV promoter, chicken β-actin promoter (CBA), CBA hybrid (CBh), chicken β-actin promoter and cytomegalovirus enhancer (CB7), chicken β-actin promoter and rabbit β-globin splicing acceptor site fusion (CAG), Rous sarcoma virus (RSV) promoter, HIV-Ltr promoter, hPGK promoter, HSV TK promoter, 7SK promoter, Mini-TK promoter, human synaptokinin I (SYN) promoter with neuron-specific expression, β-actin promoter, super core promoter 1 (SCP1), Mecp2 promoter for selective expression in neurons, minimal IL-2 promoter, Rous sarcoma virus enhancer/promoter (single), spleen focus forming virus long terminal repeat (LTR) promoter, TBG promoter, human thyroxine binding globulin gene promoter (liver specific), PGK promoter, human ubiquitin C promoter (UBC), UCOE promoter (promoter of HNRPA2B1-CBX3), synthetic CAG promoter, histone H2 promoter, histone H3 promoter, U1a1 small nuclear RNA promoter (226 nt), U1a1 small nuclear RNA promoter (226 nt), U1b2 small nuclear RNA promoter (246 nt) 26, GUSB promoter, CBh promoter, rhodopsin (Rho) promoter, silencing spleen focus forming virus (SFFV) promoter, human H1 promoter (H1), POL1 promoter, TTR minimal enhancer/promoter, b-kinesin promoter, mouse mammary tumor virus long terminal repeat sequence (LTR) promoter, human eukaryotic initiation factor 4A (EIF4A1) promoter, ROSA26 promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, tRNA promoter, and truncated versions and sequence variants of the foregoing. In a specific embodiment, the pol II promoter is EF-1α, wherein the promoter enhances transfection efficiency, transcription of transgenic genes or expression of CRISPR nucleases, the proportion of expression-positive clones, and the copy number of episomal vectors in long-term culture.

Non-limiting examples of Pol III promoters operably linked to polynucleotides encoding gRNA variants disclosed herein include, but are not limited to: U6, mini-U6, U6 truncated promoter, 7SK and H1 variants, BiH1 (bidirectional H1 promoter), BiU6, Bi7SK, BiH1 (bidirectional U6, 7SK and H1 promoter), gorilla U6, rhesus monkey U6, human 7SK, human H1 promoter, and truncated versions and sequence variants thereof. In the aforementioned embodiments, the Pol III promoter enhances transcription of the gRNA. In a specific embodiment, the Pol III promoter is U6, wherein the promoter enhances expression of the gRNA. Experimental details and data regarding the use of such promoters are provided in the Examples.

The selection of appropriate vectors and promoters is well within the skill of the art as it relates to controlled expression. The expression vector may also contain a ribosome binding site for initiation of translation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include a nucleotide sequence encoding a protein tag (e.g., a 6×His tag, a hemagglutinin tag, a fluorescent protein, etc.) that may be fused to a long-term suppressor fusion protein to generate a chimeric protein for purification or detection.

The recombinant expression vectors of the present disclosure may also include elements that promote the stable expression of the proteins and gRNAs of the present disclosure. For example, the recombinant expression vectors may include one or more of a polyadenylation signal (polyA), an intron sequence, or a post-transcriptional regulatory element such as a woodchuck hepatitis post-transcriptional regulatory element (WPRE). Exemplary polyA sequences include hGH polyA signal (short), HSV TK polyA signal, synthetic polyA signal, a split polyA sequence with a SphI restriction site between two 60 A stretches (SEQ ID NO: 3307), SV40 polyA signal, β-globin polyA signal, and the like. One of ordinary skill in the art will be able to select suitable elements to be included in the recombinant expression vectors described herein.

Polynucleotides encoding long-term suppressor fusion proteins or gRNA sequences can be individually selected and cloned into expression vectors. The selection of appropriate vectors and promoters is well within the skill of the art as it relates to controlling expression, such as for inhibiting expression of the PCSK9 gene and/or epigenetic modification. The expression vector may also contain a ribosome binding site for translation initiation and a transcriptional terminator. The expression vector may also include appropriate sequences for amplifying expression.

Nucleic acid sequences are inserted into vectors by a variety of procedures. In general, DNA is inserted into appropriate restriction endonuclease sites using techniques known in the art. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The construction of a suitable vector containing one or more of these components employs standard ligation techniques known to those skilled in the art. These techniques are well known in the art and are described in detail in scientific and patent literature. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, a viscid, a virion, or a phage that can be conveniently subjected to a recombinant DNA procedure, and the selection of the vector will generally depend on the host cell into which it is introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the host cell genome and replicates along with the chromosome into which it has been integrated. Once introduced into a suitable host cell, the expression of the long-term suppressor fusion protein or gRNA may be determined using any nucleic acid or protein assay known in the art. For example, probes complementary to any region of the polynucleotide can be used to detect and/or quantify the presence of transcribed mRNA of a long-term suppressor fusion protein by known hybridization analysis (e.g., Northern blot analysis), amplification procedures (e.g., RT-PCR), SAGE (U.S. Patent No. 5,695,937), and array-based techniques (see, e.g., U.S. Patent Nos. 5,405,783, 5,412,087, and 5,445,934).

In some embodiments, vectors are generated for transcription of long-term suppressor fusion proteins and expression and recovery of the resulting encoding mRNA. In some embodiments, mRNA is generated by in vitro transcription (IVT) using PCR products or linearized plastid DNA templates and T7 RNA polymerase, where the plastid contains a T7 promoter. If PCR products are used, the DNA sequence encoding the candidate mRNA will be cloned into plastids containing the T7 promoter, where the plastid DNA template will be linearized and then used to perform an IVT reaction to express the mRNA. Exemplary methods for the generation of such vectors and the generation and recovery of mRNA are provided in the following examples. VIII. Particles for delivery of LTRP:gRNA systems

In another aspect, the present disclosure provides a particle composition for delivering a LTRP:gRNA system to a cell or individual for transcriptional inhibition or silencing of a PCSK9 gene. Particles contemplated within the scope of the present disclosure include, but are not limited to, nanoparticles, such as synthetic nanoparticles, polymer nanoparticles, lipid nanoparticles, viral particles, and virus-like particles. The particles of the present disclosure may encapsulate a payload, such as a gRNA variant described herein, optionally in combination with an mRNA encoding a long-term suppressor fusion protein of any of the embodiments described herein. Alternatively or additionally, for example, when a ribonucleoprotein (RNP) complex is synthesized, the particles of the present disclosure may encapsulate a payload of a gRNA variant and a long-term suppressor fusion protein. In some embodiments, the particles are synthetic nanoparticles that encapsulate a payload of gRNA variants and mRNA encoding a long-term suppressor fusion protein of any of the embodiments described herein. In some embodiments, the synthetic nanoparticles comprise biodegradable polymer nanoparticles (PNPs). In some embodiments, the materials used to produce biodegradable polymer nanoparticles (PNPs) include polylactide, poly(lactic-co-glycolic acid) (PLGA), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate) and poly(isohexyl cyanoacrylate), polyglutamine (PGA), poly(ɛ-caprolactone) (PCL), cyclodextrins, and natural polymers such as chitosan, albumin, gelatin, and alginate, which are the most commonly used polymers for synthesizing PNPs. (Production and clinical development of nanoparticles for gene delivery. Molecular Therapy-Methods & Clinical Development 3:16023; doi:10.1038 (2016)). In other embodiments, the particles are lipid nanoparticles that encapsulate gRNA variants and mRNA encoding the long-term suppressor fusion protein of any embodiment described herein, which is described more fully below. In other embodiments, the particles are lipid nanoparticles (LNPs) that encapsulate gRNA variants and mRNA encoding the long-term suppressor fusion protein of any embodiment described herein separately in different particles, which are co-formulated as a mixture for administration, which is described more fully below. In other embodiments, the particles are lipid nanoparticles that separately encapsulate the gRNA variant and the mRNA encoding the long-term suppressor fusion protein of any embodiment, and the two types of particles are administered separately. a. Lipid Nanoparticles (LNP)

In another aspect, the present disclosure provides lipid nanoparticles (LNPs) for delivering the LTRP:gRNA system disclosed herein to cells or individuals for transcriptional inhibition of PCSK9 genes. In some embodiments, the LNPs disclosed herein are tissue-specific or organ-specific (e.g., liver), have excellent biocompatibility, and can efficiently deliver the system, and thus can be effectively used for transcriptional inhibition of PCSK9 genes.

The present disclosure further provides LNP compositions and pharmaceutical compositions comprising a plurality of the LNPs described herein.

In their native form, nucleic acid polymers are generally unstable in biological fluids and cannot penetrate into the cytoplasm of target cells, so a delivery system is needed. Lipid nanoparticles (LNPs) have been shown to be suitable for protecting nucleic acids and delivering nucleic acids to tissues and cells. In addition, the use of mRNA encoding long-term suppressor fusion proteins in LNPs will eliminate the possibility of undesired genomic integration compared to DNA vectors. In addition, mRNA effectively transfects mitotic and non-mitotic cells because it exerts its function in the cytoplasmic compartment without entering the cell nucleus. Therefore, using LNPs as a delivery platform will provide the additional advantage of being able to co-deliver both mRNA encoding long-term suppressor fusion proteins and gRNA into a single LNP particle.

Thus, in various embodiments, the disclosure encompasses lipid nanoparticles and compositions that can be used to achieve a variety of purposes, including the delivery of encapsulated or conjugated (e.g., complexed) therapeutic agents (e.g., nucleic acids) to cells in vitro and in vivo. In certain embodiments, the disclosure encompasses methods of treating or preventing a disease or condition in an individual in need thereof by contacting the individual with a lipid nanoparticle that encapsulates or conjugates a suitable therapeutic agent that is complexed through various physical, chemical, or electrostatic interactions between one or more lipid components used in the composition to produce the LNP. In some embodiments, the suitable therapeutic agent comprises the LTRP:gRNA system described herein.

In certain embodiments, lipid nanoparticles are suitable for delivery of nucleic acids, including, for example, mRNAs encoding long-term suppressor fusion proteins and gRNA variants disclosed herein. In some embodiments, LNPs comprise mRNAs comprising a sequence encoding a long-term suppressor fusion protein selected from the group consisting of SEQ ID NOs: 6528 to 14626 (unmodified mRNAs) and 14627 to 22725 (N1-methyl-pseudouridine modified mRNAs) or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6528-8134, 9741-11347, 14628-16233, and 17840-19446, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 6537, 9742 to 9750, 14628, 14636, and 17841 to 17849, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 6537, 9742 to 9750, 14628, 14636, and 17841 to 17849. In some embodiments, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6531, 9744, 14630, and 17843, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6531, 9744, 14630, and 17843. In some embodiments, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529, 9742, 14628, and 17841, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529, 9742, 14628, and 17841. In some embodiments, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6530, 9743, 14629, and 17842, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6530, 9743, 14629, and 17842. In some embodiments, the LNP comprises a gRNA comprising a scaffold sequence selected from the group consisting of SEQ ID NOs: 1744 to 1746, 2948 to 2956, 2958 to 2966, and 2968 to 2974, and a linked targeting sequence complementary to the sequence of the PCSK9 gene (in the case of SEQ ID NOs: 2948 to 2956, 2958 to 2966, and 2968 to 2974, the 20 non-targeting nucleotides at the 3' end are replaced by a targeting sequence complementary to the sequence of the PCSK9 gene but retaining the chemical modification at the 3' end). In some embodiments, the LNP comprises a gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 2278 to 22803. In some embodiments, the LNP comprises a gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 2278 to 22790. In some embodiments, the disclosure provides LNPs wherein the gRNA and the mRNA encoding the long-term suppressor fusion protein are incorporated into a single LNP particle. In other embodiments, the disclosure provides LNPs wherein the gRNA and the mRNA encoding the long-term suppressor fusion protein are incorporated into separate populations of LNPs, which populations can be formulated together in different ratios for administration.

The lipid nanoparticles and lipid nanoparticle compositions disclosed herein can be used to inhibit the expression of a desired protein in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more ionizable lipids described herein, wherein the lipid nanoparticles encapsulate or conjugate with a nucleic acid expressed to produce a desired protein (e.g., a messenger RNA encoding a long-term suppressor fusion protein). In some embodiments, the lipid nanoparticles and compositions can be used to inhibit the expression of a target gene in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more cationic lipids described herein, wherein the lipid nanoparticles encapsulate or conjugate with one or more nucleic acids in the LTRP:gRNA system disclosed herein. The lipid nanoparticles and compositions of the embodiments of the present disclosure can also be used alone or in combination to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA), such as to provide effects requiring co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene repressor or enzyme and gRNA for targeting a gene).

In some embodiments, the LNPs and LNP compositions described herein include at least one cationic lipid, at least one binding lipid, at least one steroid or its derivative, at least one additional lipid, or any combination thereof. Alternatively, the lipid compositions disclosed herein may include ionizable lipids, such as ionizable cationic lipids, auxiliary lipids (usually phospholipids), cholesterol, and polyethylene glycol-lipid conjugates (PEG-lipids) to improve colloidal stability in biological environments by, for example, reducing the absorption rate of plasma proteins and forming a hydration layer on the nanoparticles. These lipid compositions can be formulated at typical molar ratios of 50:10:37-39:1.5-2.5 or 20-50:8-65:25-40:1-2.5, with variations made to adjust individual properties.

The LNPs and LNP compositions of the present disclosure are configured to protect the encapsulated payload of the systems of the present disclosure in vitro and in vivo and to deliver the encapsulated payload to tissues and cells. Various embodiments of the LNPs and LNP compositions of the present disclosure are described in further detail herein. Cationic Lipids

In some embodiments, the LNPs and LNP compositions disclosed herein include at least one cationic lipid. The term "cationic lipid" refers to a lipid species having a net positive charge. In some embodiments, the cationic lipid is an ionizable cationic lipid having a net positive charge at a selected pH (e.g., physiological pH). In some embodiments, the pKa of the ionizable cationic lipid is less than about 7, so that the LNPs and LNP compositions achieve effective encapsulation of the effective load at a relatively low pH. In some embodiments, the pKa of the cationic lipid is 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. In some embodiments, the cationic lipids may be protonated at a pH lower than the pKa of the cationic lipids, and at a pH higher than the pKa, they may be substantially neutral. LNPs and LNP compositions can be safely delivered to target organs (e.g., liver, lung, heart, spleen, and tumor) and/or target cells (hepatocytes, LSEC, heart cells, cancer cells, etc.) in vivo, and exhibit a positive charge after endocytosis to release the encapsulated payload through electrostatic interactions with cationic proteins of endosomal membranes.

Early LNP formulations using permanently cationic lipids resulted in LNPs with positive surface charges and rapid clearance by phagocytic cells, which have been shown to be toxic in vivo. By becoming ionizable cationic lipids with tertiary amines (particularly tertiary amines with pKa <7), LNPs achieve efficient encapsulation of nucleic acid polymers at low pH by electrostatically interacting with the negative charges of the phosphate backbone of mRNA, which also results in a largely neutral system at physiological pH, thereby alleviating the problems associated with permanently charged cationic lipids.

As used herein, "ionizable lipid" means an amine-containing lipid that is easily protonated, and for example, it can be a lipid whose charge state changes depending on the surrounding pH. An ionizable lipid can be protonated (positively charged) at a pH below the pKa of a cationic lipid, and at a pH above the pKa, it can be substantially neutral. In one example, an LNP can include a protonated ionizable lipid and/or an ionizable lipid that exhibits neutrality. In some embodiments, the pKa of the LNP is 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. The pKa of the LNP can affect the in vivo stability and release of the nucleic acid payload of the LNP in the target cell or organ. In some embodiments, LNPs having the aforementioned pKa range can be safely delivered to target organs (e.g., liver, lung, heart, spleen, and tumor) and/or target cells (hepatocytes, LSECs, cardiac cells, cancer cells, etc.) in vivo, and exhibit a positive charge after endocytosis to release the encapsulated payload through electrostatic interactions with cationic proteins of endosomal membranes.

Ionizable lipids are ionizable compounds that generally have lipid-like characteristics and can play a role in efficiently encapsulating nucleic acids (such as mRNA of the present disclosure) into LNPs through electrostatic interactions with nucleic acids.

Depending on the type of amine and tail groups contained in the ionizable lipid, the (i) nucleic acid encapsulation efficiency, (ii) PDI (polydispersity index) and/or (iii) nucleic acid delivery efficiency of LNP to tissues and/or cells constituting an organ (e.g., hepatocytes or hepatic sinus endothelial cells in the liver) may vary. In certain embodiments, the ionizable lipid is an ionizable cationic lipid and accounts for about 46 mol% to about 66 mol% of the total lipid present in the particle.

LNPs comprising ionizable amine-containing lipids may have one or more of the following characteristics: (1) the ability to efficiently encapsulate nucleic acids; (2) the uniform size of the prepared particles (or having a low PDI value); and/or (3) excellent nucleic acid delivery efficiency to organs such as the liver, lungs, heart, spleen, bone marrow, and tumors, and/or cells constituting these organs (e.g., hepatocytes, LSECs, heart cells, cancer cells, etc.).

In certain embodiments, cationic lipid forms play an important role in both nucleic acid encapsulation via electrostatic interactions and intracellular release by disrupting endosomal membranes. Nucleic acid payloads are encapsulated within LNPs through ionic interactions with positively charged cationic lipids. Non-limiting examples of cationic lipid components used in the LNPs of the present disclosure are selected from DLin-MC3-DMA (4-(dimethylamino)butyric acid heptaheptacontriacont-6,9,28,31-tetraen-19-yl ester), DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), TNT (1,3,5-trioxane-2,4,6-trione), and TT (N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-trimethylamide). Non-limiting examples of auxiliary lipids used in the LNPs of the present disclosure are selected from DSPC (1,2-distearyl-sn-glycero-3-phosphocholine), POPC (2-oleyl-1-lauryl-sn-glycero-3-phosphocholine) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), DOPG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dilauryl-sn-glycero-3-phosphocholine (DLPC), sphingolipids and ceramides. Regarding the stability, circulation and size of LNPs, cholesterol and PEG-DMG ((R)-2,3-bis(octadecyloxy)propyl-1-(methoxypolyethylene glycol 2000) carbamate), PEG-DSG (1,2-distearyl-rac-glycero-3-methylpolyoxyethylene glycol 2000) or DSPE-PEG2k (1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) are the components used in the LNPs disclosed herein.

In some embodiments, the cationic lipid in the LNP of the present disclosure comprises a tertiary amine. In some embodiments, the tertiary amine comprises an alkyl chain connected to the N of the tertiary amine by an ether bond. In some embodiments, the alkyl chain comprises a C12-C30 alkyl chain having 0 to 3 double bonds. In some embodiments, the alkyl chain comprises a C16-C22 alkyl chain. In some embodiments, the alkyl chain comprises a C18 alkyl chain. Various cationic lipids and related analogs have been described in U.S. Patent Publication Nos. 20060083780, 20060240554, 20110117125, 20190336608, 20190381180, and 20200121809; U.S. Patent Nos. 5,208,036; 5,26 No. 4,618; No. 5,279,833; No. 5,283,185; No. 5,753,613; No. 5,785,992; No. 9,738,593; No. 10,106,490; No. 10,166,298; No. 10,221,127; and No. 11,219,634; and PCT Publication No. WO 96/10390, the disclosures of each of which are incorporated herein by reference in their entirety.

In some embodiments, the cationic lipids in the LNPs disclosed herein may include, for example, one or more ionizable cationic lipids, wherein the ionizable cationic lipids are dialkyl lipids. In other embodiments, the ionizable cationic lipids are trialkyl lipids.

In some embodiments, the cationic lipid in the LNP disclosed herein is selected from 1,2-dilinoleyl-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyl-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane ( DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxolane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpiperidin-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]- 1,2-Dilinoleylaminomethyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetyloxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-(N-phenanthroline)propane (DLin-MA), 1,2-Dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane ( DLin-S-DMA), 1-linoleyl-2-linoleyl-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyl-3-(N-methylpiperidinyl)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propane diol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyl-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N- (1-(2,3-dioleyloxy)propyl)-N,N,N-trimethoxyammonium (DOTMA), N,N-distearyl-N,N-dimethoxyammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethoxyammonium chloride (DOTAP), 3-(N-(N′,N′-dimethylaminoethane)-aminoformyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethyl bromide 2,3-Dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanemethane trifluoroacetate (DOSPA), dioctadecylamidoglyceryl spermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5′-(cholest-5-ene-3-β-oxy)- 3′-oxopentenyloxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N′-dioleylaminomethyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N′-dilinoleylaminomethyl-3-dimethylaminopropane (DLincarbDAP), and any combination thereof.

In some embodiments, the cationic lipid in the LNP of the present disclosure is selected from 4-(dimethylamino) butyric acid triheptadecanoate-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (1,3,5-trioxane-2,4,6-trione) (TNT), N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-trimethylamide (TT), and any combination thereof.

In some embodiments, the N/P ratio (nitrogen from cationic/ionizable lipids to phosphate from nucleic acids) in the disclosed LNPs is in the range of about 3:1 to 7:1, or about 4:1 to 6:1, or 3:1, or 4:1, or 5:1, or 6:1, or 7:1. Lipid Binding

In some embodiments, the LNP and LNP compositions disclosed herein include at least one conjugated lipid. In some embodiments, the conjugated lipid can be selected from polyethylene glycol (PEG)-lipid conjugates, polyamide (ATTA)-lipid conjugates, cation-polymer-lipid conjugates (CPL) and any combination of the foregoing. In some cases, the conjugated lipid can inhibit the aggregation of the LNP disclosed herein.

In some embodiments, the conjugated lipid of the LNP of the present disclosure comprises a pegylated lipid. The terms "polyethylene glycol (PEG)-lipid conjugate", "pegylated lipid", "lipid-PEG conjugate", "lipid-PEG", "PEG-lipid", "PEG-lipid" or "lipid-PEG" are used interchangeably herein and refer to a lipid linked to a polyethylene glycol (PEG) polymer, which is a hydrophilic polymer. The pegylated lipid contributes to the stability of the LNP and LNP compositions and reduces the aggregation of the LNP.

Since PEG-lipids can form surface lipids, the size of LNPs can be easily varied by changing the ratio of surface (PEG) lipids to core (cationic ionizable) lipids. In some embodiments, the PEG-lipids disclosed herein can be varied between about 1 mol% and 5 mol% to modify particle properties such as size, stability, and circulation time.

The lipid-PEG conjugate contributes to the particle stability of the nanoparticles in the LNP in serum and plays a role in preventing the aggregation between the nanoparticles. In addition, the lipid-PEG conjugate can protect nucleic acids, such as mRNA encoding the long-term inhibitor fusion protein disclosed herein or the gRNA disclosed herein, from degrading enzymes during the in vivo delivery of nucleic acids and enhance the in vivo stability of nucleic acids, and increase the half-life of the delivered nucleic acids encapsulated in the nanoparticles. Examples of PEG-lipid conjugates include, but are not limited to, PEG-DAG conjugates, PEG-DAA conjugates, and mixtures thereof. In certain embodiments, the PEG-lipid conjugate is selected from the group consisting of: PEG-diacylglycerol (PEG-DAG) conjugates, PEG-dialkoxypropyl (PEG-DAA) conjugates, PEG-phospholipid conjugates, PEG-ceramide (PEG-Cer) conjugates, and mixtures thereof.

In some embodiments, the PEGylated lipid of the LNP disclosed herein is selected from PEG-ceramide, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-dialkyloxypropylcarbamate, PEG-phosphatidylethanolamine, PEG-phospholipids, PEG-succinic acid diacylglycerol, and any combination thereof.

In some embodiments, the PEGylated lipid of the LNP disclosed herein is PEG-dialkoxypropyl. In some embodiments, the PEGylated lipid is selected from PEG-didecyloxypropyl (C10), PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dimaloxypropyl (C16), PEG-distearyloxypropyl (C18) and any combination thereof.

In other embodiments, the lipid-PEG conjugate of the LNP disclosed herein can be PEG conjugated to phospholipids, such as phosphatidylethanolamine (PEG-PE); PEG conjugated to ceramide (PEG-CER, ceramide-PEG conjugate, ceramide-PEG); cholesterol or PEG conjugated to its derivatives; PEG-c-DOMG; PEG-DMG; PEG-DLPE; PEG-DMPE; PEG-DPPC; PEG-DSPE (DSPE-PEG) and mixtures thereof, and for example, can be C16-PEG2000 ceramide (N-lauryl-sphingosine-1-{succinyl[methoxy(polyethylene glycol) 2000]}), DMG-PEG 2000, 14:0 PEG2000 PE.

In some embodiments, the PEGylated lipid of the LNP disclosed herein is selected from 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol, 4-O-(2′,3′-di(tetradecyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate, 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, and any combination thereof.

In some embodiments, the PEGylated lipid of the LNP disclosed herein is selected from mPEG2000-1,2-di-O-alkyl-sn3-aminomethylglycerol (PEG-C-DOMG), 1-[8′-(1,2-dimyristyl-3-propoxy)-formamido-3′,6′-dioxooctyl]aminomethyl-w-methyl-poly(ethylene glycol) (2KPEG-DMG), and any combination thereof.

In some embodiments, PEG is directly linked to the lipid of the PEGylated lipid. In other embodiments, PEG is linked to the lipid of the PEGylated lipid via a linker moiety selected from a non-ester linker moiety or an ester linker moiety. Non-limiting examples of non-ester linker moieties include amide (-C(O)NH-), amine (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof. For example, the linker may contain a carbamate linker moiety and an amide linker moiety. Non-limiting examples of ester-containing linker moieties include carbonate (—OC(O)O—), succinyl, phosphate (—O—(O)POH—O—), sulfonate, and combinations thereof.

The PEG moiety of the PEGylated lipid of the LNP of the present disclosure described herein can have an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain embodiments, the PEG moiety has an average molecular weight of about 750 daltons to about 5,000 daltons, about 1,000 daltons to about 4,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, or about 1750 daltons to about 2,000 daltons.

In some embodiments, the bound lipid (e.g., PEGylated lipid) comprises about 1 mol% to about 60 mol%, about 2 mol% to about 50 mol%, about 5 mol% to about 40 mol%, or about 5 mol% to about 20 mol% of the total lipid present in the LNP and/or LNP composition. In certain embodiments, the bound lipid comprises about 0.5 mol% to about 3 mol% of the total lipid present in the particle.

In additional embodiments, the bound lipid (e.g., PEGylated lipid) comprises at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 mol %, or ranges intermediate thereto, of the total lipid present in the LNP and/or LNP composition.

For the lipid in the lipid-PEG conjugate of the LNP disclosed herein, any lipid that can be conjugated to polyethylene glycol can be used (but not limited to), and phospholipids and/or cholesterol as other components of LNP can also be used. In some embodiments, the lipid in the lipid-PEG conjugate can be ceramide, dimyristylglycerol (DMG), succinyl-dioylglycerol (s-DAG), distearylphosphatidylcholine (DSPC), distearylphosphatidylethanolamine (DSPE) or cholesterol, but is not limited thereto.

In the lipid-PEG conjugates of the LNPs disclosed herein, PEG can be directly conjugated to the lipid or conjugated to the lipid via a linker moiety. Any linker moiety suitable for conjugating PEG to the lipid can be used, and includes, for example, non-ester linker moieties and ester-containing linker moieties. Non-ester linker moieties include amide (-C(O)NH-), amine (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof (e.g., linkers containing both carbamate linker moieties and amide linker moieties), but are not limited thereto. Ester -containing linker moieties include, for example, but are not limited to, carbonate (—OC(O)O—), succinyl, phosphate (—O—(O)POH—O—), sulfonate, and combinations thereof.

In some embodiments, the LNP and LNP compositions disclosed herein include at least one steroid or its derivative. In some embodiments, the steroid comprises cholesterol. In some embodiments, the LNP and LNP compositions comprise a cholesterol derivative selected from cholestanol, cholestanone, cholestenone, naphthalene sterol, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, and any combination of the foregoing.

In some embodiments, the steroid (e.g., cholesterol) of the LNPs of the present disclosure comprises about 1 mol% to about 60 mol%, about 2 mol% to about 50 mol%, about 5 mol% to about 40 mol%, or about 5 mol% to about 20 mol% of the total lipid present in the LNP and/or LNP composition. In other embodiments, the steroid (e.g., cholesterol) of the LNPs of the present disclosure comprises at least about 1 mol%, 2 mol%, 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol%, or an intermediate range of any of the foregoing, of the total lipid present in the LNP and/or LNP composition. Additional lipids

In some aspects, the LNP and LNP compositions disclosed herein include at least one additional lipid. In some embodiments, the additional lipid is a non-cationic lipid selected from anionic lipids, neutral lipids, or both. In some embodiments, the additional lipid comprises at least one phospholipid. In some embodiments, the phospholipid is selected from anionic phospholipids, neutral phospholipids, or both. The phospholipids of the elements of LNP and LNP compositions can play a role in covering and protecting the LNP core formed by the interaction of the cationic lipids and nucleic acids in the LNP, and can contribute to cell membrane permeation and endosome escape during intracellular delivery of nucleic acids by binding to the phospholipid bilayer of the target cell. The phospholipids that can promote the fusion of LNPs with cells may include, but are not limited to, any one of the phospholipids selected from the group described below.

In some embodiments, LNPs and LNP compositions comprise at least one phospholipid selected from, but not limited to, dioleyl-phosphatidylcholine (DPPC), distearyl-phosphatidylcholine (DSPC), dioleyl-phosphatidylethanolamine (DOPE), dioleyl-phosphatidylcholine (DOP C), dioleyl-phosphatidylglycerol (DOPG), phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), phosphatidylglycerol (POPG), dimalloy-phosphatidylethanolamine (DPPE), dimalloy-phosphatidylglycerol Oil (DPPG), dimyristoyl-phosphatidylethanolamine (DMPE), distearyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dioleyl-phosphatidylethanolamine (DEPE), stearyloleyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), phosphatidylethanolamine (PE), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1-oleoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleyl-sn-glycero-3-[phospho-L-serine] (DOPS), 1,2-dioleyl-sn-glycero-3-[phospho-L-serine], and any combination thereof. In one embodiment, LNPs containing DOPE can effectively deliver mRNA (with excellent drug delivery efficacy).

In some embodiments, the additional lipids (e.g., phospholipids) of the LNPs of the present disclosure account for about 1 mol% to about 60 mol%, about 2 mol% to about 50 mol%, about 5 mol% to about 40 mol%, or about 5 mol% to about 20 mol% of the total lipids present in the LNP and/or LNP composition. In other embodiments, the additional lipids (e.g., phospholipids) of the LNPs of the present disclosure account for at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol% of the total lipids present in the LNP and/or LNP composition, or an intermediate range of any of the foregoing.

It is understood that the total lipids present in LNPs and/or LNP compositions include a combination of cationic lipids or ionizable lipids, binding lipids (e.g., PEGylated lipids), steroids (e.g., cholesterol), and additional lipids (e.g., phospholipids).

LNPs and/or LNP compositions can be prepared by dissolving the total lipids (or a portion thereof) in an organic solvent (e.g., ethanol), followed by mixing with a payload (e.g., nucleic acid of the system) dissolved in an acidic buffer (e.g., pH 4) via a micromixer. At this pH, cationic lipids are positively charged and interact with negatively charged nucleic acid polymers. The resulting nanostructures containing nucleic acids are then converted to neutral LNPs when dialyzed against a neutral buffer, after which the organic solvent (e.g., ethanol) can be removed and the LNPs replaced in a physiologically relevant buffer. The LNPs and/or LNP compositions thus formed have different electron-dense nanostructured cores, in which cationic lipids are organized into reverse micelles around the encapsulated payload, which is opposite to the traditional double-layer liposome structure. In another embodiment, LNPs can form bubble-like structures with nucleic acids, which are located in water pockets along the non-electron dense lipid core. b. Lipid Nanoparticle Characteristics

In some embodiments, LNPs and/or LNP compositions comprise about 50 mol% to about 85 mol% cationic lipids or ionizable cationic lipids, about 0.5 mol% to about 10 mol% binding lipids (e.g., PEGylated lipids), about 0.5 mol% to about 10 mol% steroids (e.g., cholesterol), and about 5 mol% to about 50 mol% additional lipids (e.g., phospholipids). In some embodiments, LNPs and/or LNP compositions comprise about 50 mol% to about 85 mol% cationic lipids or ionizable cationic lipids, about 0.5 mol% to about 5 mol% binding lipids (e.g., PEGylated lipids), about 0.5 mol% to about 5 mol% steroids (e.g., cholesterol), and about 5 mol% to about 20 mol% additional lipids (e.g., phospholipids).

In some embodiments, the LNPs and/or LNP compositions disclosed herein comprise cationic lipid: additional lipid (e.g., phospholipid): steroid (e.g., cholesterol): conjugated lipid (e.g., PEGylated lipid) in a molar ratio of 20-50:10-30:30-60:0.5-5, in a molar ratio of 25-45:10-25:40-50:0.5-3, in a molar ratio of 25-45:10-20:40-55:0.5-3, or in a molar ratio of 25-45:10-20:40-55:1.0-1.5.

In some embodiments, the total lipid:payload ratio (mass/mass) of the LNPs and/or LNP compositions of the present disclosure is about 1 to about 100. In some embodiments, the total lipid:payload ratio is about 1 to about 50, about 2 to about 25, about 3 to about 20, about 4 to about 15, or about 5 to about 10. In some embodiments, the total lipid:payload ratio is about 5 to about 15, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or a range intermediate to any of the foregoing.

In certain embodiments, the LNPs of the present disclosure comprise a total lipid:nucleic acid mass ratio of about 5: 1 to about 15: 1. In some embodiments, the weight ratio of cationic lipid to nucleic acid contained in the LNP may be 1 to 20: 1, 1 to 15: 1, 1 to 10: 1, 5 to 20: 1, 5 to 15: 1, 5 to 10: 1, 7.5 to 20: 1, 7.5 to 15: 1, or 7.5 to 10: 1.

In some embodiments, the LNP of the present disclosure may comprise 20 to 50 parts by weight of cationic lipids, 10 to 30 parts by weight of phospholipids, 20 to 60 parts by weight (or 20 to 60 parts by weight) of cholesterol, and 0.1 to 10 parts by weight (or 0.25 to 10 parts by weight, 0.5 to 5 parts by weight) of lipid-PEG conjugates. Alternatively, the LNP may comprise 20 to 50% by weight of cationic lipids, 10 to 30% by weight of phospholipids, 20 to 60% by weight (or 30 to 60% by weight) of cholesterol, and 0.1 to 10% by weight (or 0.25 to 10% by weight, 0.5 to 5% by weight) of lipid-PEG conjugates based on the total nanoparticle weight. As another alternative, the LNP may comprise 25-50 wt% cationic lipid, 10-20 wt% phospholipid, 35-55 wt% cholesterol, and 0.1-10 wt% (or 0.25-10 wt%, 0.5-5 wt%) lipid-PEG conjugate based on the total nanoparticle weight.

In some embodiments, the average diameter of the LNPs of the present disclosure is about 20-200 nm, 20-180 nm, 20-170 nm, 20-150 nm, 20-120 nm, 20-100 nm, 20-90 nm, 30-200 nm, 30-180 nm, 30-170 nm, 30-150 nm, 30-120 nm, 30-100 nm, 30-90 nm, 40-200 nm, 40-180 nm, 40-170 nm, 40-150 nm, 40-120 nm, 40-100 nm, 40-90 nm, 40-80 nm, 40-70 nm, 50-200 nm, 50-180 nm, 50-170 nm, 50-150 nm, 50-120 nm, 50-100 nm, 170 nm, 80 to 150 nm, 80 to 120 nm, 80 to 100 nm, 80 to 90 nm, 90 to 200 nm, 90 to 180 nm, 90 to 170 nm, 90 to 150 nm, 90 to 120 nm, 90 to 100 nm, 60 to 90 nm, 70 to 200 nm, 70 to 180 nm, 70 to 170 nm, 70 to 150 nm, 70 to 120 nm, 70 to 100 nm, 70 to 90 nm, 80 to 200 nm, 80 to 180 nm, 80 to 170 nm, 80 to 150 nm, 80 to 120 nm, 80 to 100 nm, 80 to 90 nm, 90 to 200 nm, 90 to 180 nm, 90 to 170 nm, 90 to 150 nm, 90 to 120 nm or 90 to 100 nm, or ranges intermediate to any of the foregoing.

In some embodiments, the LNPs and/or LNP compositions disclosed herein have a positive charge at acidic pH and can encapsulate a payload (e.g., a therapeutic agent, such as a LTRP:gRNA system, or a polynucleotide encoding the same) through electrostatic charge generated by the negative charge of the payload (e.g., therapeutic agent). The term "encapsulation" refers to a mixture of lipids that surround a payload (e.g., a therapeutic agent) and entrap the payload (e.g., a therapeutic agent) under physiological conditions to form an LNP. As used herein, the term "encapsulation efficiency" is the amount of payload (e.g., a therapeutic agent) encapsulated by the LNP divided by the total amount of payload (e.g., a therapeutic agent) used to load the payload (e.g., a therapeutic agent) into the LNP. The encapsulation efficiency of the LNP and/or LNP composition can be 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 94% or greater, or 95% or greater. In other embodiments, the encapsulation efficiency of the LNP and/or LNP composition is about 80% to 99%, about 85% to 98%, about 88% to 95%, about 90% to 95%, or the effective load (e.g., the nucleic acid of the system) can be completely encapsulated in the lipid portion of the LNP composition, thereby protecting it from enzymatic degradation. In some embodiments, the effective load (e.g., therapeutic agent) is not substantially degraded after the LNP and/or LNP composition is exposed to a nuclease for at least about 20, 30, 45, or 60 minutes, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours at 37°C. In some embodiments, the effective load (e.g., nucleic acid of the system) is complexed with the lipid portion of the LNP and/or LNP composition. The LNP and/or LNP composition disclosed herein is non-toxic to mammals, such as humans.

The term "fully encapsulated" indicates that the payload (e.g., nucleic acid of the system) in the LNP and/or LNP composition is not significantly degraded after exposure to conditions that significantly degrade free DNA, RNA, or protein. In a fully encapsulated system, less than about 25%, more preferably less than about 10%, and most preferably less than about 5% of the payload (e.g., nucleic acid of the system) in the LNP and/or LNP composition is degraded under conditions that would degrade 100% of the non-encapsulated payload. "Fully encapsulated" also indicates that the LNP and/or LNP composition is serum stable and does not break down into its component parts after in vivo administration.

In some embodiments, the amount of LNP and/or LNP composition encapsulated with a payload (e.g., a therapeutic agent) is about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 100%. To about 95%, about 90% to about 95%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or ranges intermediate to any of the foregoing.

In some embodiments, the amount of effective load (e.g., nucleic acid) encapsulated within the LNP and/or LNP composition is about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 100%. To about 95%, about 90% to about 95%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or ranges intermediate to any of the foregoing.

In some embodiments, nucleic acids of the present disclosure, such as mRNA and/or gRNA encoding long-term suppressor fusion proteins, can be provided in a solution to be mixed with a lipid solution so that the nucleic acids can be encapsulated in lipid nanoparticles. Suitable nucleic acid solutions can be any aqueous solution containing encapsulated nucleic acids at various concentrations. For example, a suitable nucleic acid solution can contain one or more nucleic acids at a concentration of or greater than about 0.01 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.25 mg/ml, 1.5 mg/ml, 1.75 mg/ml, or 2.0 mg/ml. In some embodiments, the nucleic acid comprises an mRNA encoding a long-term suppressor fusion protein, and a suitable mRNA solution may contain a concentration of about 0.01 to 2.0 mg/ml, 0.01 to 1.5 mg/ml, 0.01 to 1.25 mg/ml, 0.01 to 1.0 mg/ml, 0.01 to 0.9 mg/ml, 0.01 to 0.8 mg/ml, 0.01 to 0.7 mg/ml, 0.01 to 0.6 mg/ml, 0.01 to 0.5 mg/ml, 0.01 to 0.4 mg/ml, 0.01 to 0.3 mg/ml, 0.01 to 0.2 mg/ml, 0.01 to 0.1 mg/ml, 0.05 to 1.0 mg/ml, 0.05 to 0.9 mg/ml, 0.05 to 0.8 mg/ml, 0.05 to 0.7 In some embodiments, the present invention relates to mRNA in the range of 0.1 to 0.6 mg/ml, 0.05 to 0.5 mg/ml, 0.05 to 0.4 mg/ml, 0.05 to 0.3 mg/ml, 0.05 to 0.2 mg/ml, 0.05 to 0.1 mg/ml, 0.1 to 1.0 mg/ml, 0.2 to 0.9 mg/ml, 0.3 to 0.8 mg/ml, 0.4 to 0.7 mg/ml or 0.5 to 0.6 mg/ml. In some embodiments, a suitable mRNA solution may contain mRNA at a concentration of up to about 5.0 mg/ml, 4.0 mg/ml, 3.0 mg/ml, 2.0 mg/ml, 1.0 mg/ml, 0.9 mg/ml, 0.8 mg/ml, 0.7 mg/ml, 0.6 mg/ml, 0.5 mg/ml, 0.4 mg/ml, 0.3 mg/ml, 0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml, 0.04 mg/ml, 0.03 mg/ml, 0.02 mg/ml, 0.01 mg/ml or 0.05 mg/ml. In some embodiments, a suitable gRNA solution may contain gRNA at a concentration of up to about 5.0 mg/ml, 4.0 mg/ml, 3.0 mg/ml, 2.0 mg/ml, 1.0 mg/ml, 0.9 mg/ml, 0.8 mg/ml, 0.7 mg/ml, 0.6 mg/ml, 0.5 mg/ml, 0.4 mg/ml, 0.3 mg/ml, 0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml, 0.04 mg/ml, 0.03 mg/ml, 0.02 mg/ml, 0.01 mg/ml, or 0.05 mg/ml.

In some embodiments, the average diameter of the LNPs may be 20 nm to 200 nm, 20 to 180 nm, 20 nm to 170 nm, 20 nm to 150 nm, 20 nm to 120 nm, 20 nm to 100 nm, 20 nm to 90 nm, 30 nm to 200 nm, 30 to 180 nm, 30 nm to 170 nm, 30 nm to 150 nm, 30 nm to 120 nm, 30 nm to 100 nm, 30 nm to 300 nm, to 90nm, 40nm to 200nm, 40 to 180nm, 40nm to 170nm, 40nm to 150nm, 40nm to 120nm, 40nm to 100nm, 40nm to 90nm, 40nm to 80nm, 40nm to 70nm, 50nm to 200nm, 50 to 180nm, 50nm to 170nm, 50nm to 150nm, 50nm to 120nm, 50nm to 10 0nm, 50nm to 90nm, 60nm to 200nm, 60 to 180nm, 60nm to 170nm, 60nm to 150nm, 60nm to 120nm, 60nm to 100nm, 60nm to 90nm, 70nm to 200nm, 70 to 180nm, 70nm to 170nm, 70nm to 150nm, 70nm to 120nm, 70nm to 100nm, 70nm to 90nm In some embodiments, the LNPs are sized to be easily introduced into liver tissue, hepatocytes and/or LSECs (hepatic sinus endothelial cells). The size of the LNPs can be set to be easily introduced into organs or tissues, including but not limited to the liver, lungs, heart, spleen, and into tumors. When the size of LNP is smaller than the above range, it may be difficult to maintain stability because the surface area of LNP is excessively increased, and thus delivery to the target tissue and/or drug effect may be reduced. LNP can specifically target liver tissue. Without wishing to be bound by theory, it is believed that one mechanism by which LNP can be used to deliver therapeutic agents is by mimicking the metabolic behavior of natural lipoproteins, and thus LNP can be effectively delivered to an individual through the lipid metabolism process carried out by the liver. During delivery of therapeutic agents to hepatocytes and/or LSECs (hepatic sinus endothelial cells), the diameter of the pore wall leading from the hepatic sinus lumen to the hepatocytes and LSECs is about 140 nm in mammals and about 100 nm in humans, and thus a therapeutic agent composition for delivering LNPs having a diameter within the above range may have excellent delivery efficiency to hepatocytes and LSECs when compared to LNPs having a diameter outside the above range.

According to one example, the LNP of the LNP composition may include cationic lipid: phospholipid: cholesterol: lipid-PEG conjugate in the above range or in a molar ratio of 20 to 50: 10 to 30: 30 to 60: 0.5 to 5, a molar ratio of 25 to 45: 10 to 25: 40 to 50: 0.5 to 3, a molar ratio of 25 to 45: 10 to 20: 40 to 55: 0.5 to 3, or a molar ratio of 25 to 45: 10 to 20: 40 to 55: 1.0 to 1.5. LNPs containing components in a molar ratio within the above range may have excellent delivery efficiency of therapeutic agents specific to cells of target organs.

In certain aspects, LNPs exhibit a positive charge under acidic pH conditions by showing a pKa of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7, and can efficiently encapsulate nucleic acids by easily forming complexes with nucleic acids through electrostatic interactions with therapeutic agents (such as nucleic acids that exhibit a negative charge). In such cases, LNPs can be effectively used as compositions for delivering therapeutic agents (such as nucleic acids) into cells or in vivo.

As used herein, "encapsulate" or "encapsulation" refers to the incorporation of a therapeutic agent for efficient delivery, i.e., by surrounding the therapeutic agent on the particle surface and/or embedding the therapeutic agent inside the particle. Encapsulation efficiency refers to the amount of therapeutic agent encapsulated in the LNP relative to the total amount of therapeutic agent used to prepare the LNP.

The nucleic acid of the composition is encapsulated in LNP, and can be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 94% or more, or 95% or more of the LNP encapsulated nucleic acid in the composition. In some embodiments, the nucleic acid of the composition is encapsulated in LNP so that between 80% and 99%, between 80% and 97%, between 80% and 95%, between 85% and 95%, between 87% and 95%, between 90% and 95%, between 91% or more and 95% or less, between 91% or more and 94% or less, more than 91% and 95% or less, between 92% and 99%, between 92% and 97%, or between 92% and 95% of the LNP encapsulated nucleic acid in the composition. In some embodiments, the mRNA and gRNA encoding the long-term suppressor fusion protein of any embodiment of the present disclosure are completely encapsulated in LNPs.

Target organs for delivery of nucleic acids by LNP include, but are not limited to, liver, lung, heart, spleen, and tumor. According to one embodiment, the LNP is liver tissue specific and has excellent biocompatibility, and can efficiently deliver nucleic acids of the composition, so it can be effectively used in related technical fields, such as lipid nanoparticle-mediated gene therapy. In a specific embodiment, the target cells for delivery of nucleic acids by LNP according to one embodiment can be in vivo liver cells and/or LSEC. In other embodiments, the present disclosure provides LNPs formulated for delivery of nucleic acids of each embodiment to ex vivo cells.

The present disclosure provides a pharmaceutical composition comprising: a plurality of LNPs comprising a nucleic acid, such as an mRNA and/or a gRNA variant encoding a long-term suppressor fusion protein as described herein; and a pharmaceutically acceptable carrier.

In certain embodiments, LNPs comprising nucleic acids have an electron-dense core.

The present disclosure provides LNPs comprising one or more nucleic acids, comprising: (a) mRNA and/or gRNA variants encoding long-term suppressor fusion proteins described herein; (b) one or more cationic lipids or ionizable cationic lipids or salts thereof, which account for about 50 mol% to about 85 mol% of the total lipids present in the LNP; (c) one or more non-cationic lipids, which account for about 13 mol% to about 49.5 mol% of the total lipids present in the LNP; and (d) one or more binding lipids that inhibit LNP aggregation, which account for about 0.5 mol% to about 2 mol% of the total lipids present in the particle. In another embodiment, the present disclosure provides LNPs comprising one or more nucleic acids, comprising: (a) mRNA and/or gRNA variants encoding a long-term suppressor fusion protein described herein; (b) one or more cationic lipids or ionizable cationic lipids or salts thereof, which constitute about 22 mol% to about 85 mol% of the total lipids present in the LNP; (c) one or more non-cationic lipids/phospholipids, which constitute about 10 mol% to about 70 mol% of the total lipids present in the LNP; (d) 15 mol% to about 50 mol% sterols, and (e) 1 mol% to about 5 mol% lipid-PEG or lipid-PEG-peptide in the particle. In certain embodiments, the long-term suppressor fusion protein mRNA and gRNA may be present in the same nucleic acid-lipid particle, or they may be present in different nucleic acid-lipid particles.

The present disclosure provides LNPs comprising one or more nucleic acids, comprising: (a) mRNA encoding a long-term inhibitor fusion protein described herein; (b) a cationic lipid or a salt thereof, which accounts for about 52 mol% to about 62 mol% of the total lipids present in the LNP; (c) a mixture of phospholipids and cholesterol or a derivative thereof, which accounts for about 36 mol% to about 47 mol% of the total lipids present in the LNP; and (d) a PEG-lipid conjugate, which accounts for about 1 mol% to about 2 mol% of the total lipids present in the LNP. In certain embodiments, the formulation is a four-component system comprising about 1.4 mol% PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 57.1 mol% cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 7.1 mol% DPPC (or DSPC), and about 34.3 mol% cholesterol (or its derivatives). In some embodiments, the LNP comprises mRNA and gRNA encoding CasX described herein.

In other embodiments, the LNP comprising one or more nucleic acids comprises: (a) mRNA and/or gRNA encoding a long-term suppressor fusion protein of any embodiment described herein; (b) a cationic lipid or a salt thereof, which accounts for about 46.5 mol% to about 66.5 mol% of the total lipid present in the LNP; (c) cholesterol or a derivative thereof, which accounts for about 31.5 mol% to about 42.5 mol% of the total lipid present in the LNP; and (d) a PEG-lipid conjugate, which accounts for about 1 mol% to about 2 mol% of the total lipid present in the LNP. In certain embodiments, the formulation is a three-component system that is phospholipid-free and comprises about 1.5 mol% PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 61.5 mol% cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, and about 36.9 mol% cholesterol (or its derivatives). In some embodiments, the LNP comprises mRNA and gRNA encoding CasX described herein.

Additional formulations are described in PCT Publication No. WO 2009/127060 and U.S. Patent Publication Nos. US 2011/0071208 A1 and US 2011/0076335 A1, the disclosures of which are incorporated herein by reference in their entirety.

In other embodiments, LNPs comprising one or more nucleic acids comprise: (a) mRNA and gRNA encoding a long-term suppressor fusion protein of any embodiment described herein; (b) one or more cationic lipids or ionizable cationic lipids or salts thereof, which account for about 2 mol% to about 50 mol% of the total lipids present in the LNP; (c) one or more non-cationic lipids or ionizable cationic lipids or salts thereof, which account for about 5 mol% to about 90 mol% of the total lipids present in the LNP; and (d) one or more binding lipids that inhibit particle aggregation, which account for about 0.5 mol% to about 20 mol% of the total lipids present in the LNP. In some embodiments, LNPs comprise mRNA and gRNA encoding CasX described herein.

In other embodiments, the LNP comprising one or more nucleic acids comprises: (a) mRNA and gRNA encoding a long-term suppressor fusion protein of any embodiment described herein; (b) a cationic lipid or a salt thereof, which accounts for about 30 mol% to about 50 mol% of the total lipids present in the LNP; (c) a mixture of phospholipids and cholesterol or a derivative thereof, which accounts for about 47 mol% to about 69 mol% of the total lipids present in the LNP; and (d) a PEG-lipid conjugate, which accounts for about 1 mol% to about 3 mol% of the total lipids present in the LNP. In certain embodiments, the formulation is a four-component system comprising about 2 mol% PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 40 mol% cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 10 mol% DPPC (or DSPC), and about 48 mol% cholesterol (or its derivatives). In some embodiments, the LNP comprises mRNA and gRNA encoding CasX described herein.

In other embodiments, the LNP comprising one or more nucleic acids comprises: (a) mRNA and gRNA encoding a long-term suppressor fusion protein of any embodiment described herein; (b) one or more cationic lipids or ionizable cationic lipids or salts thereof, which account for about 50 mol% to about 65 mol% of the total lipids present in the LNP; (c) one or more non-cationic lipids or ionizable cationic lipids or salts thereof, which account for about 25 mol% to about 45 mol% of the total lipids present in the LNP; and (d) one or more binding lipids that inhibit particle aggregation, which account for about 5 mol% to about 10 mol% of the total lipids present in the LNP.

In other embodiments, LNPs comprising one or more nucleic acids comprise: (a) mRNA and gRNA encoding a long-term suppressor fusion protein of any embodiment described herein; (b) a cationic lipid or a salt thereof, which accounts for about 50 mol% to about 60 mol% of the total lipids present in the LNP; (c) a mixture of phospholipids and cholesterol or a derivative thereof, which accounts for about 35 mol% to about 45 mol% of the total lipids present in the LNP; and (d) a PEG-lipid conjugate, which accounts for about 5 mol% to about 10 mol% of the total lipids present in the LNP. In some embodiments, the LNP comprises mRNA and gRNA encoding CasX described herein.

In certain embodiments, the non-cationic lipid mixture in the formulation comprises: (i) about 10 mol% to about 70 mol% of phospholipids of the total lipids present in the LNP; (ii) about 15 mol% to about 50 mol% of cholesterol or its derivatives of the total lipids present in the LNP; and 1 to 5% lipid-PEG or lipid-PEG-peptide. In a specific embodiment, the formulation is a four-component system comprising about 7 mol% PEG-lipid conjugate (e.g., PEG750-C-DMA), about 54 mol% cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 7 mol% DPPC (or DSPC), and about 32 mol% cholesterol (or its derivatives).

In other embodiments, the LNP comprising one or more nucleic acids comprises: (a) mRNA and/or gRNA encoding a long-term suppressor fusion protein of any embodiment described herein; (b) a cationic lipid or a salt thereof, which accounts for about 55 mol% to about 65 mol% of the total lipid present in the LNP; (c) cholesterol or a derivative thereof, which accounts for about 30 mol% to about 40 mol% of the total lipid present in the LNP; and (d) a PEG-lipid conjugate, which accounts for about 5 mol% to about 10 mol% of the total lipid present in the LNP. In certain embodiments, the formulation is a three-component system that is phospholipid-free and comprises about 7 mol% PEG-lipid conjugate (e.g., PEG750-C-DMA), about 58 mol% cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, and about 35 mol% cholesterol (or its derivatives). In some embodiments, the LNP comprises mRNA and gRNA encoding CasX described herein.

In other embodiments, the LNP comprising one or more nucleic acids comprises: (a) mRNA and/or gRNA encoding a long-term suppressor fusion protein of any embodiment described herein; (b) a cationic lipid or a salt thereof, which accounts for about 48 mol% to about 62 mol% of the total lipids present in the LNP; (c) a mixture of phospholipids and cholesterol or a derivative thereof, wherein the phospholipids account for about 7 mol% to about 17 mol% of the total lipids present in the LNP, and wherein cholesterol or a derivative thereof accounts for about 25 mol% to about 40 mol% of the total lipids present in the LNP; and (d) a PEG-lipid conjugate, which accounts for about 0.5 mol% to about 3.0 mol% of the total lipids present in the LNP. In some embodiments, the LNP comprises mRNA and gRNA encoding CasX described herein. IX. Methods for inhibiting PCSK9 target nucleic acid

In another aspect, the disclosure is about a method for inhibiting or silencing the transcription of a target nucleic acid sequence of a PCSK9 gene in a cell population. In some embodiments, the method comprises contacting a cell of a population with a LTRP:gRNA system disclosed herein in vitro, in vitro, or in vivo in an individual. The programmable nature of the system provided herein allows precise targeting to achieve a desired effect at one or more predetermined regions of interest in the target nucleic acid of a PCSK9 gene. In some embodiments, it may be desirable to inhibit or silence a gene that contains a mutation that causes a PCSK9-related disease or condition in an individual.

In some embodiments, the method comprises introducing into a cell a long-term suppressor fusion protein disclosed herein and one or more gRNAs having a targeting sequence complementary to a target nucleic acid, wherein the long-term suppressor fusion protein is capable of complexing with the gRNA to form RNPs, and wherein the RNPs are capable of binding to the target nucleic acid and inhibiting or silencing transcription of a PCSK9 gene in the cell (it will be understood that additional cytokines may be recruited and participate in the inhibition). In some embodiments, the method comprises introducing into a cell an mRNA encoding a long-term fusion suppressor protein disclosed herein and one or more gRNAs having a targeting sequence complementary to a PCSK9 target nucleic acid, wherein the long-term suppressor fusion protein is expressed, is capable of complexing with the gRNA to form RNPs, and wherein the RNPs are capable of binding to the target nucleic acid and inhibiting or silencing transcription of a PCSK9 gene in the cell. In some embodiments, the mRNA encoding the long-term suppressor fusion protein and the gRNA can be co-formulated in a nanoparticle for delivery to a population of cells. In some embodiments, the mRNA encoding the long-term suppressor fusion protein and the gRNA can be formulated in separate nanoparticles for delivery to a population of cells. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP), as described herein.

In some embodiments of gene transcription inhibition methods, the LTRP: gRNA system disclosed herein can be designed to target any region or vicinity of the PCSK9 gene or gene region for which transcription inhibition is sought. When the entire PCSK9 gene is to be inhibited, the disclosure contemplates the use of a guide having a targeting sequence complementary to a sequence covering or near a transcription start site (TSS). The core promoter serves as a binding platform for the transcriptional machinery, which includes Pol II and its associated general transcription factor (GTF) (Haberle, V. et al. Eukaryotic core promoters and the functional basis of transcription initiation (Nat Rev Mol Cell Biol. 19(10):621 (2018)). It has been proposed that the variability of TSS selection involves DNA "coiling" and "anti-coiling", which is marked by: (i) forward and reverse movement of the leading edge of RNA polymerase but not the trailing edge relative to DNA, and (ii) expansion and contraction of the transcription bubble. In some embodiments, the targeting sequence of the gRNA of the LTRP:gRNA system is located at one of the transcription start sites (TSS) in the PCSK9 gene. kb. In some embodiments of the method, the targeting sequence of the gRNA of the LTRP:gRNA system is complementary to a target nucleic acid sequence located within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb or 1.5 kb upstream of the TSS of the PCSK9 gene. In some embodiments of the method, the targeting sequence of the gRNA of the LTRP:gRNA system is complementary to a target nucleic acid sequence located within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb or 1.5 kb downstream of the TSS of the PCSK9 gene. In some embodiments of the method, the targeting sequence of the gRNA of the LTRP:gRNA system is complementary to a target nucleic acid sequence located within 700 bp upstream to 700 bp downstream of the TSS of the PCSK9 gene. bp, 500 bp upstream to 500 bp downstream, 300 bp upstream to 300 bp downstream, or 100 bp upstream to 100 bp downstream. In some embodiments of the method, the targeting sequence of the gRNA of the LTRP:gRNA system is complementary to a target nucleic acid sequence located within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, or 1 kb of the enhancer of the PCSK9 gene. In some embodiments of the method, the targeting sequence of the gRNA of the LTRP:gRNA system is complementary to a target nucleic acid sequence located within 1 kb of the 3' to 5' non-translated region of the PCSK9 gene. kb. In some embodiments of the method, the targeting sequence of the gRNA of the LTRP:gRNA system complements the target nucleic acid sequence located in the open reading frame of the PCSK9 gene. In some embodiments of the method, the targeting sequence of the gRNA of the LTRP:gRNA system complements the target nucleic acid sequence of the exon of the PCSK9 gene. In a specific embodiment, the targeting sequence of the gRNA of the system disclosed herein complements the target nucleic acid sequence of exon 1 of the PCSK9 gene. In other embodiments of the method, the targeting sequence of the gRNA of the system disclosed herein complements the target nucleic acid sequence of the intron of the PCSK9 gene. In other embodiments of the method, the targeting sequence of the gRNA of the system disclosed herein complements the target nucleic acid sequence of the intron-exon junction of the PCSK9 gene. In other embodiments of the method, the targeting sequence of the gRNA of the system disclosed herein is complementary to the target nucleic acid sequence of the regulatory element of the PCSK9 gene. In other embodiments of the method, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of the intergenic region of the PCSK9 gene. In other embodiments of the method, the targeting sequence of the gRNA of the system disclosed herein is complementary to the junction of the exon, intron or regulatory element of the PCSK9 gene. In the case where the targeting sequence and the regulatory element are complementary, these regulatory elements include but are not limited to the promoter region, the enhancer region, the intergenic region, the 5' non-translated region (5'UTR), the 3' non-translated region (3'UTR), the conservative element and the region containing the cis-regulatory element. In some embodiments of the method, the targeting sequence of the gRNA of the system disclosed herein is complementary to the gene target nucleic acid sequence within 1 kb of the enhancer of the PCSK9 gene. In some embodiments of the method, the targeting sequence of the gRNA of the system disclosed herein is complementary to the gene target nucleic acid sequence within the 3' non-translated region of the PCSK9 gene. The promoter region is intended to cover nucleotides within 5 kb of the start point of the coding sequence, or in the case of a gene enhancer element or a conservative element, it can be thousands of bp, hundreds of thousands of bp, or even millions of bp away from the coding sequence of the PCSK9 gene. In the foregoing, the target is one in which the target's coding PCSK9 gene is intended to be inhibited so that the gene product is not expressed or expressed at a lower level in the cell. In some embodiments, after the RNP of the system disclosed herein binds to the binding site of the target nucleic acid, the system can inhibit the transcription of the PCSK9 gene 5' to the binding site of the RNP. In other embodiments, after the RNP of the system binds to the binding site of the target nucleic acid, the system can inhibit the transcription of the PCSK9 gene 3' to the binding site of the RNP.

The present disclosure provides methods for transcriptionally inhibiting or silencing a PCSK9 target gene in a cell population. In some embodiments, the method for transcriptionally inhibiting or silencing a PCSK 9 gene comprises contacting a cell population with a LTRP:gRNA system comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 14626 (unmodified mRNAs) and 14628 to 22725 (N1-methyl-pseudouridine-modified mRNAs), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the method comprises contacting a cell population with a LTRP:gRNA system comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529-8134, 9742-11347, 14628-16233, and 17840-19446, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the method comprises contacting a cell population with a LTRP:gRNA system comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529-8134, 9742-11347, 14628-16233, and 17840-19446. In some embodiments, the method comprises contacting a cell population with a LTRP:gRNA system comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529-6537 and 9742-9750, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the method comprises contacting a cell population with a LTRP:gRNA system comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 6537 and 9742 to 9750. In some embodiments, the mRNA comprises a 5' cap sequence linked to the 5' UTR of the mRNA sequence encoding the LTRP, optionally wherein the 5' cap has a nucleic acid sequence selected from the group consisting of m7G(5')ppp(5')mAG, m7G(5')ppp(5')A, G(5')ppp(5')A, and G(5')ppp(5')G. In some embodiments, the method comprises contacting a cell population with a LTRP:gRNA system comprising a gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 1744 to 1746 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, and the gRNA comprises a linked targeting sequence that is complementary to the target nucleic acid of the PCSK9 gene. In some embodiments of the method, the LTRP: gRNA system comprises a gRNA variant comprising a sequence of SEQ ID NO: 1744 and a linked targeting sequence. In some embodiments of the method, the LTRP: gRNA system comprises a gRNA comprising a sequence of SEQ ID NO: 1745 and a linked targeting sequence. In some embodiments, the targeting sequence of the gRNA is selected from SEQ ID NO: In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1824 to 2544, 2672, 2675, 2694, and 2714. In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1824 to 1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714. In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1834, 1849, 1853, 1855, 1856, 1857, 1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875. In some embodiments, the targeting sequence is selected from the group consisting of SEQ ID NOs: The group formed by 1855, 1867 and 1869.

In some embodiments of the method of transcriptionally inhibiting or silencing a PCSK9 target gene in a cell population, the LTRP:gRNA system comprises a gRNA variant comprising the sequence SEQ ID NO: 1746 and a linked targeting sequence. In some embodiments of the method, the LTRP:gRNA system comprises a gRNA variant comprising one or more chemically modified gRNA variants, including a gRNA variant comprising a sequence selected from the group consisting of SEQ ID NO: 2948 to 2956, 2958 to 2966, and 2968 to 2976 and a targeting sequence complementary to a PCSK9 target nucleic acid that replaces 20 nucleotides on the 3' end of the gRNA of the listed sequence. In some embodiments of the method, the LTRP:gRNA system comprises a gRNA variant comprising a sequence of SEQ ID NO: 1746 and having a chemical modification, which optionally comprises a sequence selected from the group consisting of SEQ ID NOs: 2968 to 2976, and a targeting sequence complementary to the PCSK9 target nucleic acid 20 nucleotides on the 3' end of the gRNA replacing the listed sequence. In one embodiment of the method, the LTRP:gRNA system comprises a gRNA variant comprising a sequence of SEQ ID NO: 1746 and having a chemical modification, which optionally comprises a sequence of SEQ ID NO: 2968, and a targeting sequence complementary to the PCSK9 target nucleic acid 20 nucleotides on the 3' end of the gRNA replacing the listed sequence. In some embodiments, the modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 22788 to 22803. In some embodiments, the modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 22788-22790.

In some embodiments, the method for transcriptionally inhibiting or silencing the PCSK9 gene in a cell population comprises contacting the cell with a LNP comprising a gRNA variant and an mRNA encoding a long-term suppressor fusion protein. In some embodiments of the method, the LNP comprises an mRNA sequence selected from the group consisting of SEQ ID NOs: 6529 to 14626 and 14628 to 22725 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments of the method, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 14626 and 14628 to 22725. In some embodiments, the method comprises contacting a cell population with an LNP comprising an mRNA selected from the group consisting of SEQ ID NOs: 6529-8134, 9742-11347, 14628-16233, and 17840-19446, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the method comprises contacting a cell population with an LNP comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529-8134, 9742-11347, 14628-16233, and 17840-19446. In some embodiments, the method comprises contacting a cell population with an LNP comprising an mRNA selected from the group consisting of SEQ ID NOs: 6529 to 6537 and 9742 to 9750, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the method comprises contacting a cell population with an LNP comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 6537 and 9742 to 9750. In some embodiments, the mRNA further comprises a 5' cap sequence linked to the 5'UTR of the mRNA sequence encoding the LTRP, optionally wherein the 5' cap has a nucleic acid sequence selected from the group consisting of: m7G(5')ppp(5')mAG, m7G(5')ppp(5'(A, G(5')ppp(5')A, and G(5')ppp(5')G. In some embodiments, the mRNA comprises a 5'UTR, a 3'UTR, and/or a poly(A) sequence. In some embodiments of the method, the LNP comprises a gRNA comprising a sequence selected from SEQ ID NO: 1744 to 1746 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto, and the gRNA comprises a targeting sequence complementary to the target nucleic acid of the PCSK9 gene. In some embodiments of the method, the LNP comprises a gRNA variant 174 (SEQ ID NO: 1744) with a linked targeting sequence. In some embodiments of the method, the LNP comprises a gRNA comprising a sequence comprising SEQ ID NO: 1745 and a linked targeting sequence. In some embodiments of the method, the LNP comprises a gRNA comprising a sequence comprising SEQ ID NO: 1746 and a linked targeting sequence. In some embodiments of the method, the LNP comprises a chemically modified gRNA variant, which optionally comprises SEQ ID NO: 2948 to 2956, 2958 to 2966 and 2968 to 2976, and a targeting sequence that is complementary to the PCSK9 target nucleic acid of 20 nucleotides at the 3' end of the gRNA that replaces the listed sequence. In some embodiments of the method, the LNP comprises a gRNA with a chemical modification, which comprises a sequence selected from the group consisting of SEQ ID NO: 2968 to 2976, and a targeting sequence that is complementary to the target nucleic acid of 20 nucleotides at the 3' end of the gRNA that replaces the listed sequence. In a specific embodiment of the method, the LNP comprises a gRNA with a chemical modification, which comprises a sequence of SEQ ID NO: 2968 and a targeting sequence that is complementary to the target nucleic acid of 20 nucleotides at the 3' end of the gRNA that replaces the listed sequence. In some embodiments, the targeting sequence of the gRNA is selected from SEQ ID NO: In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1824 to 2544, 2672, 2675, 2694, and 2714. In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1824 to 1879, 1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714. In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1834, 1849, 1853, 1855, 1856, 1857, 1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875. In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1855, 1867, and 1869. In some embodiments of the method, the LNP comprises a chemically modified gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 22788 to 22803. In some embodiments, the LNP comprises a chemically modified gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 22788 to 22790.

In some embodiments of the method for transcriptional inhibition or silencing of a PCSK9 gene, a cell population is contacted with the LTRP:gRNA system of the embodiments such that at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% or more of the cells have their PCSK9 target nucleic acid inhibited. In some embodiments of the method, the PCSK9 gene in the cells of the population is inhibited or silenced such that the expression of the PCSK9 protein is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to cells in which the PCSK9 gene has not been targeted. In some embodiments of the method, the inhibition of the population of cells occurs in vitro. In some embodiments of the method, the inhibition of the population of cells occurs ex vivo. In some embodiments of the method, the cells are selected from the group consisting of rodent cells, mouse cells, rat cells, primate cells, and non-human primate cells. In some embodiments of the method, the cells are human cells.

In some embodiments of the method, the inhibition occurs in vitro within the cell before the cell is introduced into the individual. In some embodiments, the cell is autologous or allogeneic to the individual. In some embodiments of the method, the inhibition of the cell occurs in vivo in the individual. The systems and methods described herein can be used in various disease-related cells, such as liver cells, intestinal cells, kidney cells, central nervous system cells, smooth muscle cells, macrophages, or arterial wall cells, in which the PCSK9 gene is to be inhibited or silenced. Thus, the method can be used for application in individuals suffering from a PCSK9-related disorder, such as, but not limited to, somatic dominant hypercholesterolemia (ADH), hypercholesterolemia, elevated total cholesterol levels, hyperlipidemia, elevated low-density lipoprotein (LDL) levels, elevated LDL-cholesterol levels, decreased high-density lipoprotein levels, hepatic steatosis, coronary heart disease, ischemia, stroke, peripheral vascular disease, thrombosis, type 2 diabetes, hypertension, atherosclerosis, obesity, Alzheimer's disease, neurodegeneration, age-related macular degeneration (AMD), or a combination thereof.

The present disclosure provides a method for reversing inhibition caused by a LTRP:gRNA system. In some embodiments of the method, inhibition can be reversed by using a DNMT inhibitor. In some embodiments of the method, inhibition can be reversed by using a cytidine analog inhibitor of DNMT. In some embodiments, inhibition can be reversed by using an inhibitor selected from the group consisting of: azacytidine, decitabine, clofarabine, and zebularine. In some embodiments of the method, wherein reversal of inhibition occurs in an individual treated with the system of the present disclosure, the method comprises administering a therapeutically effective amount of a DNMT inhibitor. X. Treatment Methods

In another aspect, the present invention relates to methods for treating PCSK9-related diseases or disorders in individuals in need thereof using the LTRP:gRNA system disclosed herein. The present disclosure provides methods for treating PCSK9-related disorders in individuals in need thereof, including but not limited to somatic dominant hypercholesterolemia (ADH), hypercholesterolemia, elevated total cholesterol levels, elevated low-density lipoprotein (LDL) levels, reduced high-density lipoprotein levels, hepatic steatosis, atherosclerotic cardiovascular disease and coronary artery disease, ischemia, stroke, peripheral vascular disease, thrombosis, type 2 diabetes, hypertension, obesity, Alzheimer's disease, neurodegeneration, age-related macular degeneration (AMD), or a combination thereof. In some embodiments, the methods disclosed herein can prevent, treat and/or alleviate PCSK9-related disorders in an individual by administering a therapeutically effective amount of the composition disclosed herein to the individual. In some embodiments, the composition administered to the individual further comprises a pharmaceutically acceptable carrier, diluent or excipient.

The methods described herein can be used to treat a variety of cells associated with a disease in an individual (e.g., liver cells, intestinal cells, kidney cells, central nervous system cells, smooth muscle cells, macrophages, or arterial wall cells) in which the PCSK9 gene is to be inhibited or silenced.

In some embodiments, it may be desirable to reduce or eliminate the expression of a gene for a PCSK9 protein in an individual comprising a mutation, such as a dominant mutation that causes hypercholesterolemia or familial or autosomal dominant hypercholesterolemia. In some cases, one or both alleles of the PCSK9 gene of the individual comprise a mutation. In some cases, the PCSK9-related disease or disorder mutation is a gain-of-function mutation, including but not limited to a mutation encoding an amino acid substitution selected from the group consisting of: S127R, D129G, F216L, D374H, and D374Y relative to the sequence of SEQ ID NO: 1823. In other cases, the PCSK9-associated disease mutation is a loss-of-function mutation, including but not limited to a mutation encoding an amino acid substitution selected from the group consisting of: R46L, G106R, Y142X, N157K, R237W, and C679X relative to the sequence of SEQ ID NO: 1823. In some embodiments, it may be desirable to reduce or eliminate the expression of PCSK9 protein in an individual with elevated cholesterol levels that are not the result of a PCSK9 gene mutation.

In some embodiments, the present disclosure provides a method for treating a PCSK9-related disease or condition in an individual in need thereof, comprising inhibiting or silencing a PCSK9 gene in cells of the individual, the method comprising contacting the cells with a therapeutically effective amount of: i) an LTRP:gRNA system comprising a long-term suppressor fusion protein of any embodiment described herein and a gRNA; ii) a nucleic acid encoding a long-term suppressor fusion protein of any embodiment described herein and a gRNA; iii) an LNP or synthetic nanoparticle comprising a gRNA of any embodiment described herein and an mRNA encoding a long-term suppressor fusion protein; iv) an LNP or synthetic nanoparticle comprising a gRNA and an LNP or synthetic nanoparticle comprising an mRNA encoding a long-term suppressor fusion protein of any embodiment described herein; or v) A combination of two or more of i) to iv), wherein the target nucleic acid sequence of the cell targeted by the gRNA is inhibited or silenced by the long-term suppressor fusion protein.

In some embodiments, the present disclosure provides a method of treating a PCSK9-related disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a LTRP:gRNA system, wherein the LTRP:gRNA system comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 14626 and 14628 to 22725, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the present disclosure provides a method of treating a PCSK9-related disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a LTRP:gRNA system comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 14626 and 14628 to 22725. In some embodiments, the method comprises administering a therapeutically effective amount of a LTRP:gRNA system comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529-8134, 9741-11347, 14628-16233, and 17840-19446, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the method comprises administering a therapeutically effective dose of a LTRP:gRNA system comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529-8134, 9741-11347, 14628-16233, and 17840-19446. In some embodiments, the method comprises administering a therapeutically effective amount of a LTRP:gRNA system comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 6537 and 9742 to 9750, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the method comprises administering a therapeutically effective amount of a LTRP:gRNA system comprising an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 6537 and 9742 to 9750. In some embodiments, the mRNA further comprises a 5' cap sequence linked to the 5' UTR of the mRNA sequence encoding the LTRP, wherein the 5' cap has a nucleic acid sequence selected from the group consisting of m7G(5')ppp(5')mAG, m7G(5')ppp(5'(A, G(5')ppp(5')A, and G(5')ppp(5')G. In some embodiments, the mRNA comprises a 5' UTR, a 3' UTR, poly A, or a combination thereof.

In some embodiments of the method of treating a PCSK9-related disease or condition in an individual in need thereof, the method comprises administering a therapeutically effective amount of a LTRP:gRNA system comprising: a gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 1744 to 1746 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, and the gRNA comprises a linked targeting sequence that is complementary to a target nucleic acid of a PCSK9 gene. In some embodiments of the method, the LTRP:gRNA system comprises a gRNA variant comprising a sequence of SEQ ID NO: 1744 and a linked targeting sequence. In some embodiments of the method, the LNP comprises a gRNA variant comprising a sequence of SEQ ID NO: 1745 and a linked targeting sequence. In some embodiments of the method, the LTRP:gRNA system comprises a gRNA variant comprising a sequence of SEQ ID NO: 1746 and a linked targeting sequence. In some embodiments of the method, the LNP comprises a gRNA variant with a chemical modification, optionally comprising a sequence selected from the group consisting of SEQ ID NOs: 2948 to 2956, 2958 to 2966, and 2968 to 2976, and a targeting sequence complementary to a PCSK9 target nucleic acid replacing 20 nucleotides on the 3' end of the gRNA of the listed sequence. In some embodiments of the method, the LTRP:gRNA system comprises a gRNA variant comprising a sequence of SEQ ID NO: 1746 with a chemical modification, which optionally comprises a sequence selected from the group consisting of SEQ ID NOs: 2968 to 2976, and a targeting sequence complementary to a PCSK9 target nucleic acid 20 nucleotides from the 3' end of the gRNA replacing the listed sequence. In some embodiments of the method, the LTRP:gRNA system comprises a gRNA variant comprising a sequence of SEQ ID NO: 1746 with a chemical modification, which comprises a sequence of SEQ ID NO: 2968, and a targeting sequence complementary to a PCSK9 target nucleic acid 20 nucleotides from the 3' end of the gRNA replacing the listed sequence. In some embodiments, the chemically modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 22788 to 22803. In some embodiments of the method, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1824-2544, 2672, 2675, 2694, and 2714. In some embodiments of the method, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1824-1879, 1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714. In some embodiments of the method, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1834, 1849, 1853, 1855, 1856, 1857, 1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875. In some embodiments of the method, the targeting sequence is selected from the group consisting of SEQ ID NOs: 1855, 1867, and 1869. In some embodiments, the chemically modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 22788 to 22790.

In some embodiments of the method of treating a PCSK9-related disease or condition in an individual in need thereof, the method comprises administering a therapeutically effective amount of an LNP comprising an LTRP:gRNA system comprising a gRNA having a linked targeting sequence complementary to a target nucleic acid of a PCSK9 gene and an mRNA encoding a long-term suppressor fusion protein. In some embodiments of the method, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 14626 and 14628 to 22725 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments of the method, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 14626 and 14628 to 22725. In some embodiments of the method, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 8134, 9741 to 11347, 14628 to 16233, and 17840 to 19446, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments of the method, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 8134, 9741 to 11347, 14628 to 16233, and 17840 to 19446. In some embodiments of the method, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 6537 and 9742 to 9750, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments of the method, the LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 6529 to 6537 and 9742 to 9750. In some embodiments, the mRNA further comprises a 5' cap sequence linked to the 5' UTR of the mRNA sequence encoding the LTRP, wherein the 5' cap has a nucleic acid sequence selected from the group consisting of: m7G(5')ppp(5')mAG, m7G(5')ppp(5'(A, G(5')ppp(5')A and G(5')ppp(5')G. In some embodiments, the mRNA comprises a 5' UTR, a 3' UTR, poly A or a combination thereof.

In some embodiments, the method for transcriptional inhibition or silencing of the PCSK9 gene comprises administering to an individual a therapeutically effective dose of LNP, the LNP comprising a LTRP: gRNA system, the LTRP: gRNA system comprising: a gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 1744 to 1746 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto, and the gRNA comprises a linked targeting sequence that is complementary to the target nucleic acid of the PCSK9 gene. In some embodiments of the method, the LNP comprises a gRNA variant comprising a sequence of SEQ ID NO: 1744 and a linked targeting sequence. In some embodiments of the method, the LNP comprises a gRNA variant comprising a sequence of SEQ ID NO: 1745 and a linked targeting sequence. In some embodiments of the method, the LNP comprises a gRNA variant comprising a sequence of SEQ ID NO: 1746 and a linked targeting sequence. In some embodiments of the method, the LNP comprises a gRNA variant with a chemical modification, optionally comprising a sequence selected from the group consisting of SEQ ID NOs: 2948 to 2956, 2958 to 2966, and 2968 to 2976, and a targeting sequence complementary to a PCSK9 target nucleic acid replacing 20 nucleotides on the 3' end of the gRNA of the listed sequence. In some embodiments of the method, the LNP comprises a chemically modified gRNA variant 316 comprising a sequence selected from the group consisting of SEQ ID NOs: 2968 to 2976 and a targeting sequence complementary to the PCSK9 target nucleic acid of 20 nucleotides at the 3' end of the gRNA replacing the listed sequence. In one embodiment of the method, the LNP comprises a chemically modified gRNA variant comprising a sequence of SEQ ID NO: 2968 and a targeting sequence complementary to the PCSK9 target nucleic acid of 20 nucleotides at the 3' end of the gRNA replacing the listed sequence. In some embodiments of the method, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1824 to 2544, 2672, 2675, 2694 and 2714. In some embodiments of the method, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1824 to 1879, 1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714. In some embodiments of the method, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOs: 1834, 1849, 1853, 1855, 1856, 1857, 1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875. In some embodiments of the method, the targeting sequence is selected from the group consisting of SEQ ID NOs: 1855, 1867, and 1869. In some embodiments, the LNP comprises a modified gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 22788 to 22803. In some embodiments, the LNP comprises a modified gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 22788 to 22790.

In some embodiments of the method for treating a PCSK9-related disease or condition in an individual in need thereof, the method results in transcriptional inhibition or silencing of the PCSK9 gene in at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% or more of the cells of the targeted organ. In some embodiments of the method, the PCSK9 gene in the cells of the targeted organ is inhibited so that the expression of the PCSK9 protein is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% compared to untreated cells. The cells of the treated individual can be selected from the group consisting of rodent cells, mouse cells, rat cells, primate cells, and non-human primate cells. In some embodiments, the eukaryotic cells of the treated individual are human cells. In some embodiments, the cells are cells involved in the production of LDL, including but not limited to liver cells, or intestinal cells, kidney cells, central nervous system cells, smooth muscle cells, macrophages, retinal cells, or cells of the arterial wall (such as endothelium). In some embodiments, the cells are eye cells. In some embodiments of the methods of treating a PCSK9-related disorder in a subject, the subject is selected from the group consisting of a mouse, a rat, a pig, a non-human primate, and a human.

A variety of treatment strategies can be used in methods for treating individuals with PCSK9-related disorders. In some embodiments, the disclosure provides methods for treating individuals with PCSK9-related disorders, the methods comprising administering a LTRP:gRNA composition to the individual according to a treatment regimen comprising one or more consecutive doses. In some embodiments of the treatment regimen, the composition of the therapeutically effective dose is administered in a single dose. In other embodiments of the treatment regimen, the therapeutically effective dose is administered to the individual in two or more doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. In some embodiments of the treatment regimen, the effective amount is administered by a route selected from the group consisting of intravenous, intraportal injection, intraperitoneal, intramuscular, subcutaneous, intraocular, and oral routes.

In some embodiments of the methods of treatment, administration of a therapeutically effective amount of a LTRP:gRNA modality (including LNPs comprising a gRNA and an mRNA encoding a long-term suppressor fusion protein) to an individual suffering from a PCSK9-associated disease or disorder results in prevention or alleviation of the underlying PCSK9-associated disorder or disorder, such that an improvement is observed in the individual, notwithstanding that the individual may still suffer from the underlying disorder. In some embodiments, administration of a therapeutically effective amount of a LTRP:gRNA modality results in improvement in at least one clinically relevant endpoint, including but not limited to: change from baseline in LDL-cholesterol, reduction in plaque atheroma volume, reduction in coronary plaque, reduction in atherosclerotic cardiovascular disease (ASCVD), cardiovascular death, non-fatal myocardial infarction, ischemic stroke, non-fatal stroke, coronary revascularization, unstable angina, or vision. In some embodiments, administration of a therapeutically effective amount of a LTRP:gRNA modality results in improvement in at least two clinically relevant endpoints. In some embodiments, the subject is selected from mouse, rat, pig, dog, non-human primate, and human.

In some embodiments, the treatment method further comprises administering a chemotherapeutic agent, wherein the agent is effective in reducing LDL levels. Such agents include, but are not limited to, statins, niacin, cellulose esters, or anti-PCSK9 antibody drugs. In some embodiments, the method of treating a PCSK9-related disease or condition in an individual comprises pre-treating the individual with a therapeutic agent that increases liver LDL receptor (LDLR) expression. In some embodiments, the therapeutic agent is a PCSK9 inhibitor, such as a monoclonal antibody, a nucleic acid-based agent, or a small molecule. Exemplary therapeutic agents include, but are not limited to, evolocumab, inclisiran, alirocumab, or MK-0616. Without wishing to be bound by theory or mechanism, it is believed that pretreatment with a PCSK9 inhibitor may result in increased hepatic LDL receptor (LDLR) expression, which in turn may promote the uptake of LNPs containing a CasX:gRNA composition subsequently administered to an individual. By increasing hepatocyte uptake of LNPs, it is expected that editing of the PCSK9 gene will be enhanced so that improvements in PCSK9-related disorders may be achieved.

Methods for obtaining samples (such as body fluids or tissues) from treated individuals for analysis to determine the effectiveness of treatment and methods for preparing samples to allow analysis are well known to those skilled in the art. Methods for analysis of RNA and protein levels are discussed above and are well known to those skilled in the art. The effect of treatment can also be assessed by measuring biomarkers associated with target gene expression in the aforementioned fluids, tissues or organs collected from animals contacted with one or more compounds of the present disclosure using routine clinical methods known in the art. Biomarkers for PCSK9 disorder include, but are not limited to, PCSK9 levels, low density lipoprotein (LDL-cholesterol), lipoprotein B, non-HDL cholesterol, triglycerides and lipoprotein a, soluble CD40 ligand, osteopontin (OPN), osteoprotegerin (OPG), matrix metalloproteinases (MMPs) and myeloperoxidase (MPOP), wherein the concentrations of the markers are compared to known physiologically normal concentrations or concentrations in individuals without PCSK9 disorder.

Several mouse models exist that express mutant forms of PCSK9 and are suitable for evaluating therapeutic approaches. Transgenic mouse models of PCSK9-related disorders include knock-in mouse models with hPCSK9 (Carreras, A. In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model. MC Biology 17:4 (2019); Herbert B. et al. Increased secretion of lipoproteins in transgenic mice expressing human D374Y PCSK9 under physiological genetic control. Arterioscler Thromb Vasc Biol. 30(7):1333 (2010)). XI. Compositions, Pharmaceutical Compositions, Kits and Articles

In some embodiments, the present disclosure provides a composition comprising: i) a long-term suppressor fusion protein of the present disclosure and a gRNA comprising a targeting sequence complementary to the sequence of the PCSK9 gene; ii) one or more nucleic acids encoding the long-term suppressor fusion protein of i) and the gRNA; iii) an LNP or synthetic nanoparticle comprising a gRNA and an mRNA encoding the long-term suppressor fusion protein, or iv) an LNP or synthetic nanoparticle comprising a gRNA and an LNP or synthetic nanoparticle comprising an mRNA encoding the long-term suppressor fusion protein.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising: i) a long-term suppressor fusion protein of any embodiment of the present disclosure and a gRNA comprising a targeting sequence complementary to the sequence of the PCSK9 gene; ii) one or more nucleic acids encoding the long-term suppressor fusion protein of i) and the gRNA; iii) an LNP or synthetic nanoparticle comprising a gRNA and an mRNA encoding the long-term suppressor fusion protein, or iv) an LNP or synthetic nanoparticle comprising a gRNA and an mRNA encoding the long-term suppressor fusion protein and an LNP or synthetic nanoparticle comprising one or more pharmaceutically suitable excipients, buffers, diluents, or carriers. In some embodiments, the pharmaceutical composition is formulated for a route of administration selected from the group consisting of intravenous, intraportal injection, intraperitoneal, intramuscular, subcutaneous, intraocular, and oral routes. In one embodiment, the pharmaceutical composition is in liquid form or frozen form. In another embodiment, the pharmaceutical composition is in a prefilled syringe for a single injection. In another embodiment, the pharmaceutical composition is in solid form, for example, the pharmaceutical composition is lyophilized.

Excipients may include salts, isotonic agents, serum proteins, buffers or other pH control agents, antioxidants, thickeners, uncharged polymers, preservatives or cryoprotectants. Excipients used in the compositions disclosed herein may further include isotonic agents and buffers or other pH control agents. Such excipients may be added to achieve a preferred pH range (about 6.0 to 8.0) and volume osmotic concentration (about 50 to 400 mmol/L). Examples of suitable buffers are acetates, borates, carbonates, citrates, phosphates and sulfonated organic molecule buffers. Such buffers may be present in the composition at a concentration of 0.01 to 1.0% (w/v). The isotonic agent may be selected from any of the reagents known in the art, such as mannitol, dextrose, glucose, and sodium chloride, or other electrolytes. In some embodiments, the isotonic agent may be glucose or sodium chloride. The amount of the isotonic agent may be such that the composition has an osmotic pressure that is the same as or similar to the osmotic pressure of the biological environment into which the composition is introduced. The concentration of the isotonic agent in the composition will depend on the nature of the particular isotonic agent used and may be in the range of about 0.1% to 10%. When glucose is used, it is preferably used at a concentration of 1 to 5% w/v, more particularly 5% w/v. When the isotonic agent is sodium chloride, it is preferably used in an amount of up to 1% w/v, especially 0.9% w/v. The composition disclosed herein may further contain a preservative. Examples of preservatives include polyhexamethylene-biguanide, benzalkonium chloride, stable oxychloride complexes (such as the complex called Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, para-hydroxybenzoate and thimerosal. Typically, these preservatives are present in a concentration of about 0.001 to 1.0%. In addition, the composition disclosed herein may also contain a low temperature preservative. Preferred low-temperature preservatives are glucose, sucrose, mannitol, lactose, trehalose, sorbitol, colloidal silicon dioxide, polydextrose with a molecular weight preferably less than 100,000 g/mol, glycerol, and polyethylene glycol with a molecular weight less than 100,000 g/mol, or mixtures thereof. Glucose, trehalose, and polyethylene glycol are the most preferred. Typically, these low-temperature preservatives are present in a concentration of about 0.01% to 10%.

Additional pharmaceutical formulations suitable for administration are suitable for use in the methods and compositions disclosed herein (see, e.g., Remington's Pharmaceutical Sciences (1990) 18th edition, Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (2023) 23rd edition, Elsevier Publishing; The Merck Index (1996) 12th edition, Merck Publishing Group, Whitehouse, N.J.; and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993)).

In other embodiments, kits are provided herein, comprising the LTRP:gRNA systems, polynucleotides, vectors, and LNP formulations described herein. In other embodiments, kits are provided herein, comprising a long-term suppressor fusion protein and one or more CasX gRNAs of any embodiment of the disclosure comprising a targeting sequence complementary to the sequence of the PCSK9 gene. In certain embodiments, kits are provided herein, comprising an mRNA encoding a long-term suppressor fusion protein and one or more gRNAs of any embodiment of the disclosure comprising a targeting sequence complementary to the sequence of the PCSK9 gene target nucleic acid sequence.

In some embodiments, provided herein are kits comprising an LNP formulation encapsulating an mRNA encoding a long-term suppressor fusion protein and one or more gRNAs of any embodiment of the present disclosure comprising a targeting sequence complementary to the sequence of the PCSK9 gene. In some embodiments, the mRNA further comprises a 5' cap sequence linked to the 5' UTR of the mRNA sequence, wherein the 5' cap has a nucleic acid sequence selected from the group consisting of: m7G(5')ppp(5')mAG, m7G(5')ppp(5'(A, G(5')ppp(5')A and G(5')ppp(5')G. In some embodiments, the kit comprises a first LNP formulation comprising an mRNA and a second LNP formulation comprising a gRNA. In some embodiments, the mRNA and the gRNA are encapsulated in a single LNP formulation.

In an exemplary embodiment, the kit of the present disclosure comprises an mRNA comprising a sequence encoding a long-term suppressor fusion protein selected from the group consisting of SEQ ID NOs: 6528 to 14626 and 14627 to 22725; and a gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 1744 to 1746 and 2947 to 2976 and a linked targeting sequence complementary to a target nucleic acid in a PCSK9 gene. In some embodiments, the targeting sequence is selected from the group consisting of SEQ ID NOs: 1824 to 1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714. In some embodiments, the kits of the present disclosure comprise an mRNA comprising a sequence encoding a long-term suppressor fusion protein selected from the group consisting of SEQ ID NOs: 6528 to 14626 and 14627 to 22725; and a modified gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 2278 to 22803. In some embodiments, the kits of the present disclosure comprise an mRNA comprising a sequence encoding a long-term suppressor fusion protein selected from the group consisting of SEQ ID NOs: 6528 to 14626 and 14627 to 22725; and a modified gRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 2278 to 22790.

In some embodiments, the kit comprises a vector comprising a sequence encoding a long-term suppressor fusion protein and a gRNA disclosed in the code book.

In some embodiments, the kit further comprises a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label display reagent, instructions for use, or any combination thereof. In some embodiments, the kit further comprises a pharmaceutically acceptable carrier, diluent, or formulation.

In some embodiments, the kit further comprises a suitable container (e.g., a tube, a vial, or a tray).

In some embodiments, the kit comprises appropriate control compositions for gene suppression applications, and instructions for use.

In some embodiments, the disclosure provides compositions for treating individuals with PCSK9-related diseases. In some embodiments, the disclosure provides compositions of long-term inhibitor fusion proteins and gRNAs for use in the manufacture of medicaments for treating diseases in individuals. In some embodiments, the disclosure provides LNP compositions comprising long-term inhibitor fusion proteins and gRNAs for use in the manufacture of medicaments for treating diseases in individuals.

The following examples are illustrative only and are not intended to limit any aspect of the present disclosure in any way. Examples Example 1 : Evaluation of spacers for suppression of the PCSK9 locus in human hepatocytes when paired with LTRP5-ADD molecules

Experiments were performed to demonstrate that multiple spacers with TTC recognition motifs can induce persistent repression of therapeutically relevant endogenous loci in human cells when paired with an ADD domain-containing LTRP molecule in configuration 5 (LTRP5; schematically illustrated in Figure 1). Specifically, initial proof-of-concept experiments were performed in human Huh7 cells to evaluate a subset of spacers that exhibit sequence conservation to the non-human primate (NHP) genome to identify leading spacers for testing in future in vivo NHP studies. Materials and Methods:

Computational selection of PCSK9 targeting spacers for experimental testing with LTRP5 molecules containing the ADD domain:

To identify potential LTRP-specific spacers throughout the human PCSK9 locus, a target search region was defined starting 5 KB upstream of the transcription start site (TSS) and ending 5 KB downstream of the transcription stop site. Spacers were identified based on the availability of TTC PAMs; thus, a total of 1,121 TTC spacers were identified throughout the targeted PCSK9 locus. These spacers were then functionally annotated based on their positioning by overlapping key genomic features, i.e., determining whether the putative spacers target exons, introns, or candidate cis-regulatory elements (cCREs) within the promoter region, and/or overlap with common sites of genetic variation (e.g., SNPs). In order to narrow down and identify the initial set of spacers for experimental screening, a set of filtering criteria was applied to the extracted spacers. First, non-specific spacers were excluded by removing spacers containing up to one off-target site with a base pair mismatch with the on-target site. In addition, spacers containing the following single nucleotide repeat sequences were excluded: thymine nucleotide repeat sequences longer than four base pairs (bp), or adenine, guanine, or cytosine nucleotide repeat sequences longer than 5 bp. Then, from this filtering set, spacers with more than one off-target site containing a mismatch in the last four nucleotides of the spacer were excluded. Finally, spacers that were targeted >2 KB upstream of the TSS and >2 KB downstream of the transcriptional stop site were excluded. This resulted in a filtered set of 722 TTC spacers (SEQ ID NOs: 1824 to 2545). From this filtered set of 722 spacers, TSS-proximal (within 1100 bp upstream and downstream of the TSS) spacers were selected for experimental evaluation, resulting in the identification of 67 TTC spacers. Two additional spacers, TG-06-354 and TG-06-352, were located outside the 1100 bp critical window and were also selected for inclusion. The sequences of the resulting 69 TTC spacers are shown in Table 11. Table 11 : RNA sequences of 69 TTC spacers targeting the human PCSK9 locus . Bold spacers are those with sequence consensus between human and non-human primate genomes and were evaluated in this example. Spacer ID Spacer RNA sequence SEQ ID NO: TG-06-342 AAUUACAGGCAACAGGAAGG 1824 TG-06-343 CCCCAUGUAAGAGAGGAAGU 1825 TG-06-344 CAGUUUCUGCCUCGCCGCGG 1826 TG-06-345 GCCUCGCCGCGGCACAGGUG 1827 TG-06-346 CCCACCUGUGCCGCGGCGAG 1828 TG-06-347 CUCCUUCACCCACCUGUGCC 1829 TG-06-348 AGGCAUUCACUCCUUCACCC 1830 TG-06-349 CUGUGCCUGGUGCAGUUCCC 1831 TG-06-350 GUGUCAUAAAGAAAUUGCCU 1832 TG-06-351 UUAUGACACAGAACUCAUGC 1833 TG-06-001 GAGGAGGACGGCCUGGCCGA 1834 TG-06-002 ACCGCUGCGCCAAGGUGCGG 1835 TG-06-004 GCCAGGCCGUCCUCCUCGGA 1836 TG-06-005 GUGCUCGGGUGCUUCGGCCA 1837 TG-06-117 ACUUUGUUUGCAAAGACCUC 1838 TG-06-118 GAGUGAAAUGGCCUGCUCUG 1839 TG-06-119 GAGCAGGCCAUUUCACUCGG 1840 TG-06-120 CUCGGAAUCUGCUGUGCAUC 1841 TG-06-121 GGAAGGGCUGUCGAUACUGG 1842 TG-06-123 UCCCAGUAUCGACAGCCCUU 1843 TG-06-124 CAGUAUCGACAGCCCUUCCA 1844 TG-06-125 AGAAAGAGCAAGCCUCAUGU 1845 TG-06-128 AGAAAUCAACUGGACAAGCA 1846 TG-06-131 UGAACAUGGUGUGUAAAAGG 1847 TG-06-132 AGAAGAUUCAAUUUGCAAAG 1848 TG-06-133 AUGGUAGGCACAAGCUCAGC 1849 TG-06-134 GAAUUCUAUGGUAGGCACAA 1850 TG-06-135 GGAAAGCUGAGCUUGUGCCU 1851 TG-06-138 AGGGAUUUAUACUACAAAGA 1852 TG-06-139 AGGAGCAGCUAGUUGGUAAG 1853 TG-06-140 AAACUUAGCCUGGACCCCCU 1854 TG-06-141 ACUGGCCUUAACCUGGCAGC 1855 TG-06-142 UUCCACUGGCCUUAACCUGG 1856 TG-06-143 GAAUCAAUCCUACUGUGGAC 1857 TG-06-144 GUGGGCAGCGAGGAGUCCAC 1858 TG-06-145 UGGGUCCACCUUGUCUCCUG 1859 TG-06-146 GAAGUCUCACUGGUCAGCAG 1860 TG-06-147 GUGUUUCCUGGGUCCACCUU 1861 TG-06-149 AGCCCAGUUAGGAUUUGGGA 1862 TG-06-150 UCCCUCUGCGCGUAAUCUGA 1863 TG-06-151 CUCUGCGCGUAAUCUGACGC 1864 TG-06-152 GCCUCGCCCUCCCCAAACAG 1865 TG-06-153 GUUAAUGUUUAAUCAGAUAG 1866 TG-06-154 AGGGUGUGGGUGCUUGACGC 1867 TG-06-155 GCAGCGACGUCGAGGCGCUC 1868 TG-06-157 GGGUCUGAGCCUGGAGGAGU 1869 TG-06-158 GGAGCAGGGCGCGUGAAGGG 1870 TG-06-159 GCGCGCCCCUUCACGCGCCC 1871 TG-06-160 CGCGCCCUGCUCCUGAACUU 1872 TG-06-161 GCUCCUGCACAGUCCUCCCC 1873 TG-06-167 CACUGAAUAGCGCAGCCGCA 1874 TG-06-168 GUGGGAAGGUUCGCGGGGUU 1875 TG-06-169 CGGGGUUGGGAGACCCGGAG 1876 TG-06-170 UCGGCCUCCGGGUCUCCCAA 1877 TG-06-171 CAGUACGUUCCAGGCAUUCA 1878 TG-06-172 GCUGAAACAGAUGGAAUACU 1879 TG-06-249 AAACCAAAUCGGAACCCACU 1880 HS-6-147 UGUUGCCUGUAAUUGGAAUU 1881 HS-6-149 CCUUCCUGUUGCCUGUAAUU 1882 TG-06-122 CCUUUGUUUCUUCCCAGUAU 1883 TG-06-127 UCCUCCUGCCUGGUACACAA 1884 TG-06-137 GAAUGUACCUAUAUGACGUC 1885 TG-06-188 CCCCGGCCUCCCAUCCCUAC 1886 TG-06-243 CUUGGCACGAUCUUGGGGAC 1887 TG-06-250 GAUUUGGUUUGGAAAACAUG 1888 TG-06-251 CUCCAGGCCCUCCACCCUCC 1889 HS-6-159 CACCCCGCCCCUGUCUCGGG 1890 TG-06-354 CCCCUGCCCCUUCAGCUGGU 1925 TG-06-352 UCCCUCACCAAUUACCCCUC 1910

Assessment of PCSK9 secretion levels to select PCSK9 targeting spacers with sequence conservation to non-human primate genomes:

Of the 69 TTC spacers identified, 15 spacers that exhibited sequence conservation between the human and non-human primate genomes (bold spacers in Table 11) were first tested to assess their effects on PCSK9 secretion levels.

mRNA encoding the following molecules was generated by internal in vitro transcription (IVT) using CleanCap® AG and N1-methyl-pseudouridine. The IVT reaction was then subjected to DNase digestion and on-column oligodT purification: 1) catalytically active CasX 676 (as described in Example 2), 2) dXR1 (dCasX fused to the ZIM3-KRAB domain), and 3) LTRP5-ADD-ZIM3 (LTRP5 containing the ADD domain and the ZIM3-KRAB domain) The DNA and mRNA sequences of CasX 676 are shown in Tables 19 and 20; the DNA and mRNA sequences of dXR1 are shown in Tables 12 and 13; the DNA and mRNA sequences of LTRP5-ZIM3-ADD are shown in Tables 15 and 16.

gRNAs containing NHP conserved spacers targeting the PCSK9 locus (bold spacers in Table 11) were designed and chemically synthesized using gRNA scaffold 316. In addition, B2M targeting gRNA was used as a non-targeting control, while spacer TG-06-138 (also known as spacer 6.138; SEQ ID NO: 1852) was used to pair with dXR1, and spacer TG-06-001 (also known as spacer 6.1; SEQ ID NO: 1834) was used to pair with CasX 676. In view of the efficacy of spacer TG-06-157 (also known as spacer 6.157; SEQ ID NO: 1869), which is not a NHP conserved spacer, in sustained suppression of the PCSK9 locus, the spacer was included as a positive control, which is shown in Example 2 below.

To assess PCSK9 secretion, inoculated Huh7 cells were transfected with mRNA encoding catalytically active CasX 676, dXR1, or LTRP5-ADD-ZIM3 and gRNA with scaffold 316 and a spacer targeting the B2M or PCSK9 locus. Culture supernatants were collected 6, 18, and 36 days after transfection to assess PCSK9 secretion levels by ELISA. PCSK9 secretion levels were normalized to total cell counts. As an additional control, PCSK9 secretion was also measured in culture supernatants collected from wells containing untreated naive cells.

In the second experiment, the first 16 spacers from the previous experiment were transfected into primary human hepatocytes (PHH) via liposomes using LTRP5-ADD-ZIM3 and gRNA with scaffold 316 and spacers targeting the B2M or PCSK9 loci, and supernatants were collected 6 days after liposome transfection.

Lonza batch 31 (round 1) or 271 (round 2) cells were seeded into 96-well plates and co-lipofected with high (150 ng) or medium (75 ng) doses of mRNA+gRNA using MessengerMax reagent. Cells were maintained in culture for six days and PCSK9 attenuation was assessed by secreted protein ELISA and transcript RT-qPCR for a total of three independent rounds of screening. Medium dose mRNA/gRNA lipofectamines were collected for amplicon bisulfite sequencing. Cells were propagated for six days and DNA was extracted and amplicon bisulfite sequenced for the proximal region of the PCSK9 TSS containing 34 CpG dinucleotides (approximately 200 bp upstream to approximately 150 bp downstream). Reduction was confirmed by PCSK9 transcript RT-qPCR (as described in Example 16) and supernatant PCSK9 HTRF ELISA (as described in Example 16). Total RNA was also extracted for RNA-seq to assess specificity.

Durability of inhibition was assessed in a third experiment. HuH7 were lipofected with high doses of LTRP5-ADD-ZIM3 and gRNAs with scaffold 316 and spacers targeting either the B2M or PCSK9 loci (representing the 11 most active targeting sequences in the above experiments) in 6 replicate wells, and cells were allowed to proliferate in culture every 2 weeks with PCSK9 supernatant ELISA readouts. Replicate plates split at week 1 increased n from 6 to 12 for subsequent readouts. Results:

Quantification of normalized PCSK9 secretion levels of Huh7 cells transfected with mRNA encoding catalytically active CasX 676, dXR1, or LTRP5-ADD-ZIM3 and NHP conserved gRNA targeting the PCSK9 locus at three time points is shown in Figure 2. The data show that the use of the most NHP conserved spacers with LTRP5-ADD-ZIM3 caused sustained repression until 36 days after transfection (using spacer 7.37 targeting the B2M locus) when compared to control conditions (i.e., initial untreated cells, cells treated with dXR1, and cells treated with a non-targeting control). Specifically, the use of TSS-proximal spacers TG-06-147, TG-06-167, TG-06-133, TG-06-146, and TG-06-154 with LTRP5-ADD-ZIM3 resulted in similar or further reduced levels of sustained inhibition compared to the levels of PCSK9 secretion observed using spacer 6.1 paired with CasX 676 (Figure 2). Interestingly, the use of TG-06-352 (which was positioned beyond the 1100 bp threshold window referred to herein as "TSS-proximal") also resulted in effective inhibition (Figure 2). Similar to the findings observed in Example 2, treatment with LTRP5-ADD-ZIM3 and spacer 6.157 caused a sustained inhibition of secreted PCSK9 levels, while treatment with dXR1 and spacer 6.138 caused a transient inhibition. In addition, treatment with any of the three mRNA molecules and spacer 7.37 targeting the B2M locus did not affect PCSK9 secretion (Figure 2).

Activity levels assessed by RT-qPCR and ELISA for the top 16 spacer sequences demonstrated that TG-06-154, TG-06-141, and TG-06-157 were the most active spacers (Table 17). Targeted sequence TG-06-154 showed the highest average secreted protein reduction by ELISA across rounds and doses. PCSK9 targeted spacers induced average CpG methylation of approximately 40 to 60%, with the exception of targeted spacers TG-06-146 and TG-06-142, which exhibited lower average CpG methylation (Table 17).

The specificity and durability of inhibition are quantified in Table 18. Treatment with LTRP5-ADD-ZIM3 and targeting sequences TG-06-133, TG-06-139, TG-06-158, and TG-06-160 showed an increase in the number of off-target differentially expressed genes, while targeting sequences TG-06-141, TG-06-144, TG-06-154, and TG-06-157 exhibited no more than 1 differentially expressed off-target gene. Persistence, measured by the percent change in PCSK9 reduction from week 1 to week 9, indicated that the PCSK9 targeted spacers maintained inhibition within approximately 30% of initial inhibition over 9 weeks, with the exception of the targeted spacers TG-06-001, TG-06-139, and TG-06-150 (Table 18).

These results demonstrate that delivery of mRNA encoding LTRP molecules with ADD domains with appropriate PCSK9- targeting gRNA can result in sustained repression of endogenous target loci in human cells. In addition, these experiments show that several human spacers that share common sequences with non-human primate species achieve strong phenotypic effects due to targeting therapeutically relevant loci, which supports the potential use of these selected spacers in preclinical efficacy studies using non-human primate models. Table 12 : Coding sequences of dXR1 and LTRP1-ZIM3 mRNA molecules evaluated in Experiment #1 of this Example * dXR or LTRP ID Components DNA Sequence SEQ ID NO: dXR1 (codon optimized) 5'UTR AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC 3047 START codon + NLS + linker ATGGCCCCTAAGAAGAAGCGTAAAGTGAGCCGGGGCGGCAGCGGCGGCGGCAGCGCC 3048 dCasX491 CAGGAGATTAAACGGATCAACAAGATCAGAAGAAGACTTGTGAAAGACAGCAACACCAAGAAGGCCGGCAAGACAGGCCCCATGAAAACCCTGCTGGTTTAGAGTGATGACACCCGATCTGAGAGAGCGGCTGGAAAACCTGAGAAAGAAGCCTGAAAATATCCCCCAGCCCATCAGCAATACA TCTAGAGCCAACCTGAATAAGCTGCTGACCGATTACACCGAAATGAAGAAGGCGATCCTGCATGTGTACTGGGAAGAGTTCCAGAAGGACCCTGTGGGCCTGATGAGCCGGGTGGCCCAGCCTGCCAGCAAGAAGATCGATCAGAACAAGCTGAAACCTGAGATGGACGAGAAGGGCAACCTG ACCACCGCCGGCTTTGCCTGCTCTCAGTGTGGCCAGCCCCTGTTCGTGTACAAGCTGGAGCAGGTGTCTGAGAAGGGCAAGGCTTACACCAACTTCGGACGGTGCAATGTGGCCGAGCACGAAAAGCTGATCCTGCTGGCCCAGCTGAAGCCCGAGAAGGATAGCGACGAAGCCGTGACA TATAGCCTGGGAAAGTTTGGGCAGAGGGCCCTGGATTTCTACAGCATTCATGTGACCAAGGAGTCCACCCACCCCGTGAAGCCCTGGCCCAGATCGCCGGAAACAGATACGCCTCCGGACCTGTGGGAAAGGCCCTGAGCGACGCATGTATGGGCACAATCGCCTCCTTCCTGTCTAAGTAC CAGGACATCATCGAACACCAGAAGGTGGTGAAGGGCAACCAGAAGAGACTGGAGAGCCTGCGGGAGCTGGCCGGCAAGGAAAACCTGGAATACCCTAGCGTGACCCTGCCACCTCAGCCTCACACCAAGGAGGGCGTTGATGCCTACAACGAAGTGATCGCCCGGGTGCGAATGTGGGTG AACCTGAACCTGTGGCAGAAGCTGAAGCTAAGCAGAGATGATGCCAAGCCTCTGCTGAGACTGAAGGGATTCCCTTCCTTTCCTCTGGTCGAGAGACAGGCCAACGAAGTGGACTGGTGGGACATGGTGTGTAACGTGAAGAAGCTGATCAACGAGAAAAAGGAGGATGGCAAGGTGTTTTGG CAGAATCTGGCTGGCTACAAGAGACAGGAAGCCCTGAGACCATACCTGAGCAGCGAGGAAGATCGGAAGAAGGGAAAGAAATTCGCTCGGTACCAGCTGGGCGACCTGCTGCTGCACCTGGAAAAGAAGCACGGCGAGGACTGGGGAAAGGTGTACGACGAGGCCTGGGAGCGGATTGACAAG AAAGTGGAAGGCCTGAGCAAGCACATCAAGCTGGAAGAGGAACGGAGAAGCGAGGACGCCCAGAGCAAGGCCGCCCTGACCGACTGGCTGCGGGCTAAGGCCAGCTTCGTGATCGAGGGCCTGAAGGAGGCCGACAAGGACGAGTTCTGCAGATGCGAGCTGAAGCTGCAGAAGTGGTACGGGG ACCTGCGGGGAAAGCCCTTCGCCATCGAAGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCAGTACAACTGTGCCTTCATCTGGCAGAAGGACGGCGTGAAGAAGCTGAACCTGTACCTGATCATCAACTACTTCAAGGGCGGCAAGCTGCGGTTCAAGAAGATCAAACCTGAAG CCTTCGAAGCCAACAGATTCTACACCGTGATCAACAAAAAGAGCGGCGAGATCGTGCCCATGGAGGTGAACTTCAACTTCGACGACCCCAACCTGATCATCCTGCCTCTGGCCTTTGGCAAGAGACAGGGCAGAGAATTCATCTGGAACGACCTGCTGTCCCTGGAAACCGGCAGCCTGAAGC TGGCCAACGGAAGAGTGATCGAGAAGACACTGTACAACAGAAGAACCCGGCAGGATGAGCCTGCCCTGTTCGTGGCCCTGACCTTCGAGCGGCGGGAGGTCCTGGACTCCTCCAATATCAAACCAATGAACCTGATCGGCGTGGCAAGAGGCGAAAACATCCCCGCCGTGATCGCCCTGACCG ACCCCGAGGGCTGCCCACTGAGCCGGTTTAAGGATAGCCTGGGAAACCCAACCCACATCCTGAGAATCGGCGAGAGCTATAAGGAGAAGCAGCGGACCATCCAGGCCAAGAAGGAGGTGGAGCAGCGGAGAGCCGGCGGCTACAGCCGGAAGTACGCCAGCAAAGCCAAGAATCTGGCAGACGA TATGGTGAGAAACACCGCTAGAGATCTGCTGTACTACGCCGTGACCCAGGATGCCATGCTGATCTTCGCCAACCTGAGCCGGGGCTTCGGCCGGCAGGGCAAGCGGACCTTCATGGCCGAGAGACAGTACACACGGATGGAGGACTGGCTGACCGCCAAGCTGGCCTACGAGGGCCTGAGCAA GACCTACCTGTCCAAGACACTGGCCCAGTACACCTCCAAGACATGCAGCAACTGTGGGTTTACCATCACCAGCGCCGACTACGACAGGGTGCTGGAGAAGCTGAAGAAGACAGCAACAGGCTGGATGACCACAATTAACGGCAAGGAGCTGAAGGTGGAGGGCCAGATTACCTACTACAACAG ATACAAGAGACAGAACGTAGTCAAGGACCTGTCCGTCGAGCTGGATAGACTGAGCGAAGAATCTGTGAACAACGACATCTCCTCCTGGACAAAGGGCAGAAGCGGAGAAGCTCTGAGCCTCCTGAAGAAAAGATTCTCCCATAGACCCGTGCAGGAGAAGTTCGTGTGCCTGAACTGCGGCTT CGAGACACACGCAGCCGAGCAAGCCGCCCTGAACATCGCCAGATCCTGGCTGTTCCTGCGGAGCCAGGAGTACAAGAAATACCAGACAAACAAGACAACCGGCAACACCGATAAGAGAGCCTTCGTCGAGACCTGGCAGTCCTTTACCGGAAGAAGCTTAAGGAGGTGTGGAAACCTGCCGTG 3049 Connector + Buffer Sequence CGGTCTGGCGGATCTGGCGGAGGCTCCACAAGC 3050 ZIM3-KRAB ATGAACAACTCCCAGGGCAGAGTGACCTTCGAGGACGTGACCGTGAATTTTACACAGGGAGAGTGGCAGAGACTGAACCCCGAGCAGAGAAAACCTGTACCGGGATGGTGATGCTGGAAAACTACAGCAATCTGGTGTCCGTGGGCCAGGGC GAGACCACAAAGCCTGACGTGATCCTGCGTCTGGAGCAGGGCAAGGAACCCTGGCTGGAGGAGGAGGAGGTGCTGGGAAGCGGACGGGCCGAGAAGAACGGCGACATCGGCGGACAGATCTGGAAGCCTAAGGACGTGAAAGAAAGCCTG 3051 Buffer Sequence + NLS ACCAGCCCCAAGAAAAAGAGAAAAGTC 3052 Logo GACTACAAGGATGACGATGACAAGGACTACAAGGATGACGACGACAAG 3053 STOP codon + buffer sequence TAATAGATAAGCGGCCGCTTAATTAA 3054 3'UTR GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG 3055 Buffer sequence TCTAG 3056 Poly(A) tail AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3057 LTRP1 (codon optimized) 5'UTR AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC 3047 START codon + NLS + buffer sequence + linker ATGGCCCCTAAGAAGAAGCGTAAAGTGAGCCGGGTGAACGGCAGCGGCAGCGGCGGCGGC 3058 START codon + DNMT3A catalytic domain ATGAACCACGACCAGGAGTTCGACCCCCCTAAGGTGTACCCTCCCGTCCCCGCCGAGAAGAGAAAGCCCATCCGGGTCCTGAGCCTGTTCGATGGCATCGCCACCGGTCTGCTGGTGCTGAAGGACCTGGGCATCCAGGTGGATAGGTACATTGCCTCCGAGGTGTGCGAGGACTCCATCACCGTGGGAATGGTGCGTCATCAGGGCAAGATCATGTACGTGGGCG ACGTGCGGAGCGTGACACAGAAGCATATCCAGGAGTGGGGCCCTTTCGACCTGGTGATCGGCGGCAGCCCTTGCAATGACCTGAGCATCGTGAACCCAGCCCGGAAGGGCCTGTACGAGGGAACCGGCAGACTGTTCTTCGAGTTTTACAGACTGCTGCACGACGCCCGGCCTAAGGAAGGCGACGACCGGCCCTTTCTTTTGGCTGTTCGAGAATGTGGTGGCCATG GGAGTCAGCGACAAGCGGGATATTAGCCGGTTCCTGGAGAGCAACCCCGTGATGATCGATGCCAAGGAAGTGAGCGCCGCCCACCGGGCCAGATACTTCTGGGGCAATCTGCCTGGCATGAACAGACCCCTGGCCAGCACCGTGAACGACAAGCTGGAGCTGCAGGAGTGCCTGGAGCACGGCCGGATCGCCAAGTTCAGCAAGGTGAGAACCATCACCACCCGAA GCAACAGCATCAAACAAGGCAAGGACCAGCACTTTCCTGTGTTCATGAACGAGAGGAGGACATCCTGTGGTGTACCGAGATGGAGAGAGTGTTCGGGTTCCCAGTCCACTACACAGATGTCAGCAACATGTCTAGACTGGCCAGACAGAGACTGCTGGGAAGAAGCTGGTCCGTCCCTGTGATCAGACACCTGTTCGCCCCTCTGAAGGAGTACTTCGCCTGCGTG 3059 Connector AGCAGCGGCAACAGCAACGCCAACAGCCGGGGCCCCAGCTTCTCTAGCGGCCTGGTGCCACTGTCCCTGAGAGGGAGCCAC 3060 DNMT3L interaction domain ATGGGCCCCATGGAGATCTACAAAACCGTGAGCGCCTGGAAGCGGCAGCCTGTGCGCGTGCTGAGCCTGTTTCGGAATATCGATAAAGTCCTGAAAAGCCTGGGATTCCTGGAGAGCGGCTCTGGCTCCGGCGGTGGCACCCTGAAGTACGTGGAGGATGT GACAAACGTGGTCAGACGGGATGTGGAGAAGTGGGGCCCCTTCGATCTGGTGTACGGCAGCACCCAACCCCTGGGCAGCTCTTGTGACCGGTGCCCTGGCTGGTACATGTTTCAGTTCCACCGGATCCTGCAGTACGCCCTGCCGAGACAGGAGTCCCAGC GGCCATTCTTTTGGATTTTCATGGACAACTTGCTGCTGACCGAGGATGACCAGGAAACTACCACTCGGTTCCTGCAGACCGAAGCCGTGACCCTGCAGGACGTGAGAGGCCGGGACTACCAGAACGCCATGCGGGTGTGGTCCAACATCCCTGGACTGAAA AGCAAGCACGCACCTCTGACCCCTAAAGAAGAGGAGTACCTGCAGGCCCAGGTGCGGAGCAGAAGCAAGCTGGACGCCCCTAAGGTGGATCTGCTGGTGAAGAATTGCCTCCTGCCCCTGAGAGAGTACTTCAAGTATTTCAGCCAGAATAGTCTGCCCCTG 3061 Connector GGCGGCCCAAGCAGCGGCGCCCCTCCTCCCAGCGGCGGCAGCCCAGCCGGCTCCCCAACCTCTACCGAGGAGGGCACCTCTGAGTCCGCCACCCCCGAGAGCGGCCCTGGCACCTCCACCGAGCCCAGCGAG GGCAGCGCACCCGGCAGCCCTGCCGGCAGCCCCACCTCCACAGAGGAGGGAACCAGCACCGAGCCCAGCGAAGGCAGCGCCCCAGGCACCAGCACCGAGCCTAGTGAGGGCGGCTCTGGCGGCGGCAGCGCC 3062 dCasX491 CAGGAGATTAAACGGATCAACAAGATCAGAAGAAGACTTGTGAAAGACAGCAACACCAAGAAGGCCGGCAAGACAGGCCCCATGAAAACCCTGCTGGTTTAGAGTGATGACACCCGATCTGAGAGAGCGGCTGGAAAACCTGAGAAAGAAGCCTGAAAATATCCCCCAGCCCATCAGCAATACA TCTAGAGCCAACCTGAATAAGCTGCTGACCGATTACACCGAAATGAAGAAGGCGATCCTGCATGTGTACTGGGAAGAGTTCCAGAAGGACCCTGTGGGCCTGATGAGCCGGGTGGCCCAGCCTGCCAGCAAGAAGATCGATCAGAACAAGCTGAAACCTGAGATGGACGAGAAGGGCAACCTG ACCACCGCCGGCTTTGCCTGCTCTCAGTGTGGCCAGCCCCTGTTCGTGTACAAGCTGGAGCAGGTGTCTGAGAAGGGCAAGGCTTACACCAACTTCGGACGGTGCAATGTGGCCGAGCACGAAAAGCTGATCCTGCTGGCCCAGCTGAAGCCCGAGAAGGATAGCGACGAAGCCGTGACA TATAGCCTGGGAAAGTTTGGGCAGAGGGCCCTGGATTTCTACAGCATTCATGTGACCAAGGAGTCCACCCACCCCGTGAAGCCCTGGCCCAGATCGCCGGAAACAGATACGCCTCCGGACCTGTGGGAAAGGCCCTGAGCGACGCATGTATGGGCACAATCGCCTCCTTCCTGTCTAAGTAC CAGGACATCATCGAACACCAGAAGGTGGTGAAGGGCAACCAGAAGAGACTGGAGAGCCTGCGGGAGCTGGCCGGCAAGGAAAACCTGGAATACCCTAGCGTGACCCTGCCACCTCAGCCTCACACCAAGGAGGGCGTTGATGCCTACAACGAAGTGATCGCCCGGGTGCGAATGTGGGTG AACCTGAACCTGTGGCAGAAGCTGAAGCTAAGCAGAGATGATGCCAAGCCTCTGCTGAGACTGAAGGGATTCCCTTCCTTTCCTCTGGTCGAGAGACAGGCCAACGAAGTGGACTGGTGGGACATGGTGTGTAACGTGAAGAAGCTGATCAACGAGAAAAAGGAGGATGGCAAGGTGTTTTGG CAGAATCTGGCTGGCTACAAGAGACAGGAAGCCCTGAGACCATACCTGAGCAGCGAGGAAGATCGGAAGAAGGGAAAGAAATTCGCTCGGTACCAGCTGGGCGACCTGCTGCTGCACCTGGAAAAGAAGCACGGCGAGGACTGGGGAAAGGTGTACGACGAGGCCTGGGAGCGGATTGACAAG AAAGTGGAAGGCCTGAGCAAGCACATCAAGCTGGAAGAGGAACGGAGAAGCGAGGACGCCCAGAGCAAGGCCGCCCTGACCGACTGGCTGCGGGCTAAGGCCAGCTTCGTGATCGAGGGCCTGAAGGAGGCCGACAAGGACGAGTTCTGCAGATGCGAGCTGAAGCTGCAGAAGTGGTACGGGG ACCTGCGGGGAAAGCCCTTCGCCATCGAAGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCAGTACAACTGTGCCTTCATCTGGCAGAAGGACGGCGTGAAGAAGCTGAACCTGTACCTGATCATCAACTACTTCAAGGGCGGCAAGCTGCGGTTCAAGAAGATCAAACCTGAAG CCTTCGAAGCCAACAGATTCTACACCGTGATCAACAAAAAGAGCGGCGAGATCGTGCCCATGGAGGTGAACTTCAACTTCGACGACCCCAACCTGATCATCCTGCCTCTGGCCTTTGGCAAGAGACAGGGCAGAGAATTCATCTGGAACGACCTGCTGTCCCTGGAAACCGGCAGCCTGAAGC TGGCCAACGGAAGAGTGATCGAGAAGACACTGTACAACAGAAGAACCCGGCAGGATGAGCCTGCCCTGTTCGTGGCCCTGACCTTCGAGCGGCGGGAGGTCCTGGACTCCTCCAATATCAAACCAATGAACCTGATCGGCGTGGCAAGAGGCGAAAACATCCCCGCCGTGATCGCCCTGACCG ACCCCGAGGGCTGCCCACTGAGCCGGTTTAAGGATAGCCTGGGAAACCCAACCCACATCCTGAGAATCGGCGAGAGCTATAAGGAGAAGCAGCGGACCATCCAGGCCAAGAAGGAGGTGGAGCAGCGGAGAGCCGGCGGCTACAGCCGGAAGTACGCCAGCAAAGCCAAGAATCTGGCAGACGA TATGGTGAGAAACACCGCTAGAGATCTGCTGTACTACGCCGTGACCCAGGATGCCATGCTGATCTTCGCCAACCTGAGCCGGGGCTTCGGCCGGCAGGGCAAGCGGACCTTCATGGCCGAGAGACAGTACACACGGATGGAGGACTGGCTGACCGCCAAGCTGGCCTACGAGGGCCTGAGCAA GACCTACCTGTCCAAGACACTGGCCCAGTACACCTCCAAGACATGCAGCAACTGTGGGTTTACCATCACCAGCGCCGACTACGACAGGGTGCTGGAGAAGCTGAAGAAGACAGCAACAGGCTGGATGACCACAATTAACGGCAAGGAGCTGAAGGTGGAGGGCCAGATTACCTACTACAACAG ATACAAGAGACAGAACGTAGTCAAGGACCTGTCCGTCGAGCTGGATAGACTGAGCGAAGAATCTGTGAACAACGACATCTCCTCCTGGACAAAGGGCAGAAGCGGAGAAGCTCTGAGCCTCCTGAAGAAAAGATTCTCCCATAGACCCGTGCAGGAGAAGTTCGTGTGCCTGAACTGCGGCTT CGAGACACACGCAGCCGAGCAAGCCGCCCTGAACATCGCCAGATCCTGGCTGTTCCTGCGGAGCCAGGAGTACAAGAAATACCAGACAAACAAGACAACCGGCAACACCGATAAGAGAGCCTTCGTCGAGACCTGGCAGTCCTTTACCGGAAGAAGCTTAAGGAGGTGTGGAAACCTGCCGTG 3049 Connector CGGTCTGGCGGATCTGGCGGAGGCTCCACAAGC 3050 ZIM3-KRAB ATGAACAACTCCCAGGGCAGAGTGACCTTCGAGGACGTGACCGTGAATTTTACACAGGGAGAGTGGCAGAGACTGAACCCCGAGCAGAGAAAACCTGTACCGGGATGGTGATGCTGGAAAACTACAGCAATCTGGTGTCCGTGGGCCAGGGC GAGACCACAAAGCCTGACGTGATCCTGCGTCTGGAGCAGGGCAAGGAACCCTGGCTGGAGGAGGAGGAGGTGCTGGGAAGCGGACGGGCCGAGAAGAACGGCGACATCGGCGGACAGATCTGGAAGCCTAAGGACGTGAAAGAAAGCCTG 3051 Buffer Sequence + NLS ACCAGCCCCAAGAAAAAGAGAAAAGTC 3052 Logo GACTACAAGGATGACGATGACAAGGACTACAAGGATGACGACGACAAG 3053 STOP codon + buffer sequence TAATAGATAAGCGGCCGCTTAATTAA 3054 3'UTR GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG 3055 Buffer sequence TCTAG 3056 Poly(A) tail AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3057 * The components are listed in 5' to 3' order within the construct. Table 13 : Full-length RNA sequences of dXR1 and LTRP1-ZIM3 mRNA molecules evaluated in Experiment #1 of this example . Modification ' mψ ' = N1- methyl - pseudouridine dXR or LTRP ID RNA- seq SEQ ID NO: dXR1 AAAmψAAGAGAGAAAAGAAGAGmψAAGAAGAAAmψAmψAAGAGCCACCAmψGGCCCCmψAAGAAGAAGCGmψAAAGmψGAGCCGGGGCGGCAGCGGCGGCGGCAGCGCCCAGGAGAmψmψAAACGGAmψCA ACAAGAmψCAGAAGAAGACmψmψGmψGAAAGACAGCAACACCAAGAAGGCCGGCAAGACAGGCCCCAmψGAAAACCCmψGCmψGGmψmψAGAGmψGAmψGACACCCGAmψCmψGAGAGAGCGGCmψGGAAA ACCmψGAGAAAGAAGCCmψGAAAAmψAmψCCCCCAGCCCAmψCAGCAAmψACAmψCmψAGAGCCAACCmψGAAmψAAGCmψGCmψGACCGAmψmψACACCGAAAmψGAAGAAGGCGAmψCCmψGCAmψGmψ GmψACmψGGGAAGAGmψmψCCAGAAGGACCCmψGmψGGGCCmψGAmψGAGCCGGGmψGGCCCAGCCmψGCCAGCAAGAAGAmψCGAmψCAGAACAAGCmψGAAACCmψGAGAmψGGACGAGAAGGGCAACC mψGACCACCGCCGGCmψmψmψGCCmψGCmψCmψCAGmψGmψGGCCAGCCCCmψGmψmψCGmψGmψACAAGCmψGGAGCAGGmψGmψCmψGAGAAGGGCAAGGCmψmψACACCAACmψACmψmψCGGACGGm ψGCAAmψGmψGGCCGAGCACGAAAAGCmψGAmψCCmψGCmψGGCCCAGCmψGAAGCCCGAGAAGGAmψAGCGACGAAGCCGmψGACAmψAmψAGCCmψGGGAAAGmψmψmψGGGCAGAGGGCCCmψGGAmψ mψmψCmψACAGCAmψmψCAmψGmψGACCAAGGAGmψCCACCCACCCGmψGAAGCCCCmψGGCCCAGAmψCGCCGGAAACAGAmψACGCCmψCCGGACCmψGmψGGGAAAGGCCCmψGAGCGACGCAmψGm ψAmψGGGCACAAmψCGCCmψCCmψmψCCmψGmψCmψAAGmψACCAGGACAmψCAmψCAmψCGAACACCAGAAGGmψGGmψGAAGGGCAACCAGAAGAGACmψGGAGAGCCmψGCGGGAGCmψGGCCGGCAA GGAAAACCmψGGAAmψACCCmψAGCGmψGACCCmψGCCACCmψCAGCCmψCACACCAAGGAGGGCGmψmψGAmψGCCmψACAACGAAGmψGAmψCGCCCGGGmψGCGAAmψGmψGGGmψGAACCmψGAACC mψGmψGGCAGAAGCmψGAAGCmψAAGCAGAAmψGAmψGCCAAGCCmψCmψGCmψGAGACmψGAAGGGAmψmψCCCmψmψCCmψmψmψCCmψCmψGGmψCGAGAGACAGGCCAACGAAGmψGGACmψGGmψ GGGACAmψGGmψGmψGmψAACGmψGAAGAAGCmψGAmψCAACGAGAAAAAGGAGGAmψGGCAAGGmψGmψmψmψmψGGCAGAAmψCmψGGCmψGGCmψACAAGAGACAGGAAGCCCmψGAGACCAmψACCm ψGAGCAGCGAGGAAGAmψCGGAAGAAGGGAAAGAAAmψmψCGCmψCGGmψACCAGCmψGGGCGACCmψGCmψGCmψGCACCmψGGAAAAGAAGCACGGCGAGGACmψGGGGAAAGGmψGmψACGACGAGGC CmψGGGAGCGGAmψmψGACAAGAAAGmψGGAAGGCCmψGAGCAAGCACAmψCAAGCmψGGAAGAGGAACGGAGAAGCGAGGACGCCCAGAGCAAGGCCGCCCmψGACCGACmψGGCmψGCGGGCmψAAGGC CAGCmψmψCGmψGAmψCGAGGGCCmψGAAGGAGGCCGACAAGGACGAGmψmψCmψGCAGAmψGCGAGCmψGAAGCmψGCAGAAGmψGGmψACGGGGACCmψGCGGGGAAAGCCCmψmψCGCCAmψCGAAGC CGAGAACAGCAmψCCmψGGACAmψCAGCGGCmψmψCAGCAAGCAGmψACAACmψGmψGCCmψmψCAmψCmψGGCAGAAGGACGGCGmψGAAGAAGCmψGAACCmψGmψACCmψGAmψCAmψCAACmψACmψ mψCAAGGGCGGCAAGCmψGCGGmψmψCAAGAAGAmψCAAACCmψGAAGCCmψmψCGAAGCCAACAGAmψmψCmψACACCGmψGAmψCAACAAAAAGAGCGGCGAGAmψCGmψGCCCAmψGGAGGmψGAACm ψmψCAACmψmψCGACGACCCCAACCmψGAmψCAmψCCmψGCCmψCmψGGCCmψmψmψGGCAAGAGACAGGGCAGAGAAmψmψCAmψCmψGGAACGACCmψGCmψGmψCCCmψGGAAACCGGCAGCCmψGAA GCmψGGCCAACGGAAGAGmψGAmψCGAGAAGACACmψGmψACAACAGAAGAACCCGGCAGGAmψGAGCCmψGCCCmψGmψmψCGmψGGCCCmψGACCmψmψCGAGCGGCGGGAGGmψCCmψGGACmψCCmψ CCAAmψAmψCAAACCAAmψGAACCmψGAmψCGGCGmψGGCAAGAGGCGAAAACAmψCCCCGCCmψGAmψCGCCCmψGACCGACCCCGAGGGCmψGCCCACmψGAGCCGGmψmψmψAAGGAmψAGCCmψGG GAAACCCCAACCCACAmψCCmψGAGAAmψCGGCGAGAGCmψAmψAAGGAGAAGCAGCGGACCAmψCCAGGCCAAGAAGGAGGmψGGAGCAGCGGAGCCGGCGGCmψACAGCCGGAAGmψACGCCAGCAAA GCCAAGAAmψCmψGGCAGACGAmψAmψGGmψGAGAAACACCGCmψAGAGAmψCmψGCmψGmψACmψACGCCGmψGACCCAGGAmψGCCAmψGCmψGAmψCmψmψCGCCAACCmψGAGCCGGGGCmψmψCGG CCGGCAGGGCAAGCGGACCmψmψCAmψGGCCGAGAGACAGmψACACACGGAmψGGAGGACmψGGCmψGACCGCCAAGCmψGGCCmψACGAGGGCCmψGAGCAAGACCmψACCmψGmψCCAAGACACmψGGC CCAGmψACACCmψCCAAGACAmψGCAGCAACmψGmψGGGmψmψmψACCAmψCACCAGCGCCGACmψACGACAGGGmψGCmψGGAGAAGCmψGAAGAAGACAGCAACAGGCmψGGAmψGACCACAAmψmψAA CGGCAAGGAGCmψGAAGGmψGGAGGGCCAGAmψmψACCmψACmψACAACAGAmψACAAGAGACAGAACGmψAGmψCAAGGACCmψGmψCCGmψCGAGCmψGGAmψAGACmψGAGCGAAGAAmψCmψGmψGA ACAACGACAmψCmψCCmψCCmψGGACAAAGGGCAGAAGCGGAGAAGCmψCmψGAGCCmψCCmψGAAGAAAAGAmψmψCmψCCCAmψAGACCCGmψGCAGGAGAAGmψmψCGmψGmψGCCmψGAACmψGCGG CmψmψCGAGACACACGCAGCCGAGCAAGCCGCCCmψGAACAmψCGCCAGAmψCCmψGGCmψGmψmψCCmψGCGGAGCCAGGAGmψACAAGAAAmψACCAGACAAACAAGACAACCGGCAACACCGAmψAAG AGAGCCmψmψCGmψCGAGACCmψGGCAGmψCCmψmψmψmψACCGGAAGAAGCmψmψAAGGAGGmψGmψGGAAACCmψGCCGmψGCGGmψCmψGGCGGAmψCmψGGCGGAGGCmψCCACAAGCAmψGAACAA CmψCCCAGGGCAGAGmψGACCmψmψCGAGGACGmψGACCGmψGAAmψmψmψmψACACAGGGAGAGmψGGCAGAGACmψGAACCCCGAGCAGAGAAACCmψGmψACCGGGAmψGmψGAmψGCmψGGAAAACm ψACAGCAAmψCmψGGmψGmψCCGmψGGGCCAGGGCGAGACCACAAAGCCmψGACGmψGAmψCCmψGCGmψCmψGGAGCAGGGCAAGGAACCCmψGGCmψGGAGGAGGAGGAGGmψGCmψGGGAAGCGGACG GGCCGAGAAGAACGGCGACAmψCGGCGGACAGAmψCmψGGAAGCCmψAAGGACGmψGAAAGAAAGCCmψGACCAGCCCCAAGAAAAAGAGAAAAGmψCGACmψACAAGGAmψGACGAmψGACAAGGACmψA CAAGGAmψGACGACGACAAGmψAAmψAGAmψAAGCGGCCGCmψmψAAmψmψAAGCmψGCCmψmψCmψGCGGGGCmψmψGCCmψmψCmψGGCCAmψGCCCmψmψCmψmψCmψCmψCCCmψmψGCACCmψGmψ ACCmψCmψmψGGmψCmψmψmψGAAmψAAAGCCmψGAGmψAGGAAGmψcmψagaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 3063 LTRP1 AAAmψAAGAGAGAAAAGAAGAGmψAAGAAGAAAmψAmψAAGAGCCACCAmψGGCCCCmψAAGAAGAAGCGmψAAAGmψGAGCCGGGmψGAACGGCAGCGGCAGCGGCGGCGGCAmψGAACCACGACCAGGAGmψmψCGACCCCCCmψAAGGmψGmψACCCmψCCCGmψCCCCGCCGAGAAGAGAAAGCCCAmψCCGGGmψ CCmψGAGCCmψGmψmψCGAmψGGCAmψCGCCACCGGmψCmψGCmψGGmψGCmψGAAGGACCmψGGGCAmψCCAGGmψGGAmψAGGmψACAmψmψGCCmψC CGAGGmψGmψGCGAGGACmψCCAmψCACCGmψGGGAAmψGGmψGCGmψCAmψCAGGGCAAGAmψCAmψGmψACGmψGGGCGACGmψGCGGAGCGmψGACA CAGAAGCAmψAmψCCAGGAGmψGGGGCCCmψmψmψCGACCmψGGmψGAmψCGGCGGCAGCCCmψmψGCAAmψGACCmψGAGCAmψCGmψGAACCCAGCCC GGAAGGGCCmψGmψACGAGGGAACCGGCAGACmψGmψmψCmψmψCGAGmψmψmψmψACAGACmψGCmψGCACGACGCCCGGCCmψAAGGAAGGCGACGAC CGGCCCmψmψCmψmψmψmψGGCmψGmψmψCGAGAAmψGmψGGmψGGCCAmψGGGAGmψCAGCGACAAGCGGGAmψAmψmψAGCCGGmψmψCCmψGGAGAG CAACCCCGmψGAmψGAmψCGAmψGCCAAGGAAGmψGAGCGCCGCCCACCGGGCCAGAmψACmψmψCmψGGGGCAAmψCmψGCCmψGGCAmψGAACAGACC CCmψGGCCAGCACCGmψGAACGACAAGCmψGGAGCmψGCAGGAGmψGCCmψGGAGCACGGCCGGAmψCGCCAAGmψmψCAGCAAGGmψGAGAACCAmψCA CCACCCGAAGCAACAGCAmψCAAACAAGGCAAGGACCAGCACmψmψmψCCmψGmψGmψmψCAmψGAACGAGAAGGAGGACAmψCCmψGmψGGmψGmψACC GAGAmψGGAGAGAGmψGmψmψCGGGmψmψCCCAGmψCCACmψACACAGAmψGmψCAGCAACAmψGmψCmψAGACmψGGCCAGACAGAGACmψGCmψGGGA AGAAGCmψGGmψCCGmψCCCmψGmψGAmψCAGACACCmψGmψmψCGGCCCCmψCmψGAAGGAGmψACmψmψCGCCmψGCGmψGAGCAGCGGCAACAGCAAC GCCAACAGCCGGGGCCCCAGCmψmψCmψCmψAGCGGCCmψGGmψGCCACmψGmψCCCmψGAGAGGGAGCCACAmψGGGCCCCAmψGGAGAmψCmψACAAA ACCGmψGAGCGCCmψGGAAGCGGCAGCCmψGmψGCGCGmψGCmψGAGCCmψGmψmψmψCGGAAmψAmψCGAmψAAAGmψCCmψGAAAAGCCmψGGGAmψm ψCCmψGGAGAGCGGCmψCmψGGCmψCCGGCGGmψGGCACCCmψGAAGmψACGmψGGAGGAmψGmψGACAAACGmψGGmψCAGACGGGAmψGmψGGAGAAG mψGGGGGCCCCmψmψCGAmψCmψGGmψGmψACGGCAGCACCCAACCCmψGGGCAGCmψCmψmψGmψGACCGGmψGCCCmψGGCmψGGmψACAmψGmψmψmψ CAGmψmψCCACCGGAmψCCmψGCAGmψACGCCCmψGCCGAGACAGGAGmψCCCAGCGGCCAmψmψCmψmψmψmψGGAmψmψmψmψCAmψGGACAACmψmψ GCmψGCmψGACCGAGGAmψGACCAGGAAACmψACCACmψCGGmψmψCCmψGCAGACCGAAGCCGmψGACCCmψGCAGGACGmψGAGAGGCCGGGACmψAC CAGAACGCCAmψGCGGGmψGmψGGmψCCAACAmψCCCmψGGACmψGAAAAGCAAGCACGCACCmψCmψGAACCCmψAAAGAAGAGGAGmψACCmψGCAGG CCCAGGmψGCGGAGCAGAAGCAAGCmψGGACGCCCCmψAAGGmψGGAmψCmψGCmψGGmψGAAGAAmψmψGCCmψCCmψGCCCCmψGAGAGAGmψACmψm ψCAAGmψAmψmψmψCAGCCAGAAmψAGmψCmψGCCCCmψGGGCGGCCCAAGCAGCGGCCCCmψCCmψCCCAGCGGCGGCAGCCCAGCCGGCmψCCCCAACCmψCmψACCGAGGAGGGCACCmψCmψGAGmψCCGCCACCCCCGAGAGCGGCCCmψGGCACCmψCCACCGAGCCCAGCGAGGGCAGCGCACCCGGCAGC CCmψGCCGGCAGCCCCACCmψCCACAGAGGAGGGAACCAGCACCGAGCCCAGCGAAGGCAGCGCCCCAGGCACCAGCACCGAGCCmψAGmψGAGGGCGGCmψCmψGGCGGCGGCAGCGCCCAGGAGAmψmψAAACGGAmψCAACAAGAmψCAGAAGAAGACmψmψGmψGAAAGACAGCAACACCAAGAAGGCCGGCAAGA CAGGCCCCAmψGAAAACCCmψGCmψGGmψmψAGAGmψGAmψGACACCCGAmψCmψGAGAGAGCGGCmψGGAAAACCmψGAGAAAGAAGCCmψGAAAAmψA mψCCCCCAGCCCAmψCAGCAAmψACAmψCmψAGAGCCAACCmψGAAmψAAGCmψGCmψGACCGAmψmψACACCGAAAmψGAAGAAGGCGAmψCCmψGCAm ψGmψGmψACmψGGGAAGAGmψmψCCAGAAGGACCCmψGmψGGGCCmψGAmψGAGCCGGGmψGGCCCAGCCmψGCCAGCAAGAAGAmψCGAmψCAGAACAA GCmψGAAACCmψGAGAmψGGACGAGAAGGGCAACCmψGACCACCGCCGGCmψmψmψGCCmψGCmψCmψCAGmψGmψGGCCAGCCCCmψGmψmψCGmψGmψ ACAAGCmψGGAGCAGGmψGmψCmψGAGAAGGGCAAGGCmψmψACACCAACmψACmψmψCGGACGGmψGCAAmψGmψGGCCGAGCACGAAAAGCmψGAmψC CmψGCmψGGCCCAGCmψGAAGCCCGAGAAGGAmψAGCGACGAAGCCGmψGACAmψAmψAGCCmψGGGAAAGmψmψmψGGGCAGAGGGCCCmψGGAmψmψm ψCmψACAGCAmψmψCAmψGmψGACCAAGGAGmψCCACCCACCCGmψGAAGCCCCmψGGCCCAGAmψCGCCGGAAACAGAmψACGCCmψCCGGACCmψGm ψGGGAAAGGCCCmψGAGCGACGCAmψGmψAmψGGGCACAAmψCGCCmψCCmψmψCCmψGmψCmψAAGmψACCAGGACAmψCAmψCAmψCGAACACCAGAAG GmψGGmψGAAGGGCAACCAGAAGAGACmψGGAGAGCCmψGCGGGAGCmψGGCCGGCAAGGAAAACCmψGGAAmψACCCmψAGCGmψGACCCmψGCCACCm ψCAGCCmψCACACCAAGGAGGGCGmψmψGAmψGCCmψACAACGAAGmψGAmψCGCCCGGGmψGCGAAmψGmψGGGmψGAACCmψGAACCmψGmψGGCAGA AGCmψGAAGCmψAAGCAGAmψGAmψGCCAAGCCmψCmψGCmψGAGACmψGAAGGGAmψmψCCCmψmψCCmψmψmψCCmψCmψGGmψCGAGAGACAGGC CAACGAAGmψGGACmψGGmψGGGACAmψGGmψGmψGmψAACGmψGAAGAAGCmψGAmψCAACGAGAAAAAGGAGGAmψGGCAAGGmψGmψmψmψmψGGCA GAAmψCmψGGCmψGGCmψACAAGAGACAGGAAGCCCmψGAGACCAmψACCmψGAGCAGCGAGGAAGAmψCGGAAGAAGGGAAAGAAAmψmψCGCmψCGGm ψACCAGCmψGGGCGACCmψGCmψGCmψGCACCmψGGAAAAGAAGCACGGCGAGGACmψGGGGAAAGGmψGmψACGACGAGGCCmψGGGAGCGGAmψmψGA CAAGAAAGmψGGAAGGCCmψGAGCAAGCACAmψCAAGCmψGGAAGAGGAACGGAGAAGCGAGGACGCCCAGAGCAAGGCCGCCCmψGACCGACmψGGCmψ GCGGGCmψAAGGCCAGCmψmψCGmψGAmψCGAGGGCCmψGAAGGAGGCCGACAAGGACGAGmψmψCmψGCAGAmψGCGAGCmψGAAGCmψGCAGAAGmψG GmψACGGGGACCmψGCGGGGAAAGCCCmψmψCGCCAmψCGAAGCCGAGAACAGCAmψCCmψGGACAmψCAGCGGCmψmψCAGCAAGCAGmψACAACmψGm ψGCCmψmψCAmψCmψGGCAGAAGGACGGCGmψGAAGAAGCmψGAACCmψGmψACCmψGAmψCAmψCAACmψACmψmψCAAGGGCGGCAAGCmψGCGGmψm ψCAAGAAGAmψCAAACCmψGAAGCCmψmψCGAAGCCAACAGAmψmψCmψACACCGmψGAmψCAACAAAAAGAGCGGCGAGAmψCGmψGCCCAmψGGAGGm ψGAACmψmψCAACmψmψCGACGACCCCAACCmψGAmψCAmψCCmψGCCmψCmψGGCCmψmψmψGGCAAGAGACAGGGCAGAGAAmψmψCAmψCmψGGAAC GACCmψGCmψGmψCCCmψGGAAACCGGCAGCCmψGAAGCmψGGCCAACGGAAGAGmψGAmψCGAGAAGACACmψGmψACAACAGAAGAACCCGGCAGGAm ψGAGCCmψGCCCmψGmψmψCGmψGGCCCmψGACCmψmψCGAGCGGCGGGAGGmψCCmψGGACmψCCmψCCAAmψAmψCAAACCAAmψGAACCmψGAmψCG GCGmψGGCAAGAGGCGAAAACAmψCCCCGCCmψGAmψCGCCCmψGACCGACCCCGAGGGCmψGCCCACmψGAGCCGGmψmψmψAAGGAmψAGCCmψGGGAAACCCAACCCACAmψCCmψGAGAAmψCGGCGAGAGCmψAmψAAGGAGAAGCAGCGGACCAmψCCAGGCCAAGAAGGAGGmψGGAGCAGCGGAGAGCCGGC GGCmψACAGCCGGAAGmψACGCCAGCAAAGCCAAGAAmψCmψGGCAGACGAmψAmψGGmψGAGAAACACCGCmψAGAGAmψCmψGCmψGmψACmψACGCC GmψGACCCAGGAmψGCCAmψGCmψGAmψCmψmψCGCCAACCmψGAGCCGGGGCmψmψCGGCCGGCAGGGCAAGCGGACCmψmψCAmψGGCCGAGAGACAG mψACACACGGAmψGGAGGACmψGGCmψGACCGCCAAGCmψGGCCmψACGAGGGCCmψGAGCAAGACCmψACCmψGmψCCAAGACACmψGGCCCAGmψACA CCmψCCAAGACAmψGCAGCAACmψGmψGGGmψmψmψACCAmψCACCAGCGCCGACmψACGACAGGGmψGCmψGGAGAAGCmψGAAGAAGACAGCAACAGG CmψGGAmψGACCACAAmψmψAACGGCAAGGAGCmψGAAGGmψGGAGGGCCAGAmψmψACCmψACmψACAACAGAmψACAAGAGACAGAACGmψAGmψCAA GGACCmψGmψCCGmψCGAGCmψGGAmψAGACmψGAGCGAAGAAmψCmψGmψGAACAACGACAmψCmψCCmψCCmψGGACAAAGGGCAGAAGCGGAGAAGC mψCmψGAGCCmψCCmψGAAGAAAAGAmψmψCmψCCCAmψAGACCCGmψGCAGGAGAAGmψmψCGmψGmψGCCmψGAACmψGCGGCmψmψCGAGACACACG CAGCCGAGCAAGCCGCCCmψGAACAmψCGCCAGAmψCCmψGGCmψGmψmψCCmψGCGGAGCCAGGAGmψACAAGAAAmψACCAGACAAACAAGACAACCGG CAACACCGAmψAAGAGAGCCmψmψCGmψCGAGACCmψGGCAGmψCCmψmψmψmψACCGGAAGAAGCmψmψAAGGAGGmψGmψGGAAACCmψGCCGmψGCG GmψCmψGGCGGAmψCmψGGCGGAGGCmψCCACAAGCAmψGAACAACmψCCCAGGGCAGAGmψGACCmψmψCGAGGACGmψGACCGmψGAAmψmψmψmψAC ACAGGGAAGmψGGCAGAGACmψGAACCCCGAGCAGAGAAACCmψGmψACCGGGAmψGmψGAmψGCmψGGAAAACmψACAGCAAmψCmψGGmψGmψCCGm ψGGGCCAGGGCGAGACCACAAAGCCmψGACGmψGAmψCCmψGCGmψCmψGGAGCAGGGCAAGGAACCCmψGGCmψGGAGGAGGAGGAGGmψGCmψGGGAA GCGGACGGGCCGAGAAGAACGGCGACAmψCGGCGGACAGAmψCmψGGAAGCCmψAAGGACGmψGAAAGAAAGCCmψGACCAGCCCCAAGAAAAAGAGAAA AGmψCGACmψACAAGGAmψGACGAmψGACAAGGACmψACAAGGAmψGACGACGACAAGmψAAmψAGAmψAAGCGGCCGCmψmψAAmψmψAAGCmψGCCmψ mψCmψGCGGGGCmψmψGCCmψmψCmψGGCCAmψGCCCmψmψCmψmψCmψCmψCCCmψmψGCACCmψGmψACCmψCmψmψGGmψCmψmψmψGAAmψAAAGC CmψGAGmψAGGAAGmψcmψagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 3064 Table 14 : Full-length protein sequences of dXR1 and LTRP1-ZIM3 molecules evaluated in Experiment #1 of this Example dXR or LTRP ID AA sequence SEQ ID NO: dXR1 MAPKKKRKVSRGGSGGGSAQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQK RLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQL GDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGG KLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPL SRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITS ADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAF VETWQSFYRKKLKEVWKPAVRSGGSGGGSTSMNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLTSPKKKRKV 3065 LTRP1 MAPKKKRKVSRVNGSGSGGGMNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWG PFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLAST VNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNS NANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGW YMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNC LLPLREYFKYFSQNSLPLGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGGSGGGSAQEI KRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKL KPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYAS GPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPS FPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEE ERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPE AFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVA RGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKR TFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVN NDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGST SMNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLTSPKKKRKV 3066 Table 15 : DNA and RNA sequences encoding LTRP5-ZIM3 and LTRP5-ZIM3-ADD mRNA molecules evaluated in this example * LTRP molecules Components DNA Sequence SEQ ID NO RNA- seq SEQ ID NO LTRP5-ZIM3 5'UTR AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC 3047 AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC 3115 START codon + NLS + linker ATGGCCCCTAAGAAGAAGCGTAAAGTGAGCCGG 3104 AUGGCCCCUAAGAAGAAGCGUAAAGUGAGCCGG 3116 START codon + DNMT3A catalytic domain See the sequence in Table 12 3059 AUGAACCACGACCAGGAGUUCGACCCCCUAAGGUGUACCCUCCCGUCCCCGCCGAGAAGAGAAAGCCCAUCCGGGUCCUGAGCCUGUUCGAUGGCAUCGCCACCGGUCUGCUGGUGCUGAAGGACCUGGGCAUCCAGGUGGAUAGGUACAUUGCCUCCGAGGUGUGCGAGGACUCCAUCACCGUGGGAAUGGUGCGUCAUCAGGGCAAGAUCAUGUACGUGGGCG ACGUGCGGAGCGUGACACAGAAGCAUAUCCAGGAGUGGGGCCUUUCGACCUGGUGAUCGGCGGCAGCCCUUGCAAUGACCUGAGCAUCGUGAACCCAGCCCGGAAGGGCCUGUACGAGGGAACCGGCAGACUGUUCUUCGAGUUUACAGACUGCUGCACGACGCCCGGCCUAAGGAAGGCGACGACCGGCCCUUCUUUUGGCUGUUCGAGAAUGUGGUGGCCAUG GGAGUCAGCGACAAGCGGGAUAUUAGCCGGUUCCUGGAGAGCAACCCCGUGAUGAUCGAUGCCAAGGAAGUGAGCGCCGCCCACCGGGCCAGAUACUUCUGGGGCAAUCUGCCUGGCAUGAACAGACCCCUGGCCAGCACCGUGAACGACAAGCUGGAGCUGCAGGAGUGCCUGGAGCACGGCCGGAUCGCCAAGUUCAGCAAGGUGAGAACCAUCACCACCCGAA GCAACAGCAUCAAACAAGGCAAGGACCAGCACUUUCCUGUGUUCAUGAACGAGAAGGAGGACAUCCUGGUGUACCGAGAUGGAGAGUGUUCGGGUUCCCAGUCCACUACACAGAUGUCAGCAACAUGUCUAGACUGGCCAGACAGAGACUGCUGGGAAGAAGCUGGUCCGUCCCCUGUGAUCAGACACCUGUUCGCCCUCUGAAGGAGUACUUCGCCUGCGUG 3117 Connector AGCAGCGGCAACAGCAACGCCAACAGCCGGGGCCCCAGCTTCTCTAGCGGCCTGGTGCCACTGTCCCTGAGAGGGAGCCAC 3060 AGCAGCGGCAACAGCAACGCCAACAGCCGGGGCCCCAGCUUCUCUAGCGGCCUGGGCCACUGUCCCUGAGAGGGAGCCAC 3118 DNMT3L interaction domain See the sequence in Table 12 3061 AUGGGCCCCAUGGAGAUACAAAACCGUGAGCGCCCUGGAAGCGGCAGCCUGUGCGCGUGCUGAGCCUGUUUCGGAAUAUCGAUAAAGUCCUGAAAAGCCUGGGAUUCCUGGAGAGCGGCUCUGGCUCCGGCGGUGGCACCCUGAAGUACGUGGAGGAUGU GACAAACGUGGUCAGACGGGAUGUGGAGAAGUGGGGCCCCUUCGAUCUGGUGUACGGCAGCACCCAACCCCUGGGCAGCUCUUGUGACCGGUGCCCUGGCUGGUACAUGUUUCAGUUCCACCGGAUCCUGCAGUACGCCCUGCCGAGACAGGAGUCCCAGC GGCCAUUCUUUUGGAUUUCCAUGGACAACUUGCUGCUGACCGAGGAUGACCAGGAAACUACCACUCGGUUCCUGCAGACCGAAGCCGUGACCCUGCAGGACGUGAGAGGCCGGGACUACCAGAACGCCAUGCGGGUGUGGUCCAACAUCCCUGGACUGAAA AGCAAGCACGCACCUCUGACCCCUAAAGAAGAGGAGUACCUGCAGGCCCAGGUGCGGAGCAGAAGCAAGCUGGACGCCCCUAAGGUGGAUCUGCUGGUGAAGAAUUGCCUCCUGCCCCUGAGAGAGUACUUCAAGUAUUUCAGCCAGAAUAGUCUGCCCCUG 3119 Connector GGAGGCAGCGGCGGCGGC 3105 GGAGGCAGCGGCGGCGGC 3105 ZIM3-KRAB See the sequence in Table 12 3051 AUGAACAACUCCCAGGGCAGAGUGACCUUCGAGGACGUGACCGUGAAUUUUACACAGGGAGAGUGGCAGAGACUGAACCCCGAGCAGAGAAACCUGUACCGGGAUGUGAUGCUGGAAAACUACAGCAAUCUGGUGUCCGUGGGCCAGGGC GAGACCACAAAGCCUGACGUGAUCCUGCGUCUGGAGCAGGGCAAGGAACCCUGGCUGGAGGAGGAGGAGGUGCUGGGAAGCGGACGGGCCGAAGAACGGCGACAUCGGCGGACAGAUCUGGAAGCCUAAGGACGUGAAAGAAAGCCUG 3120 Connector GGCGGCCCAAGCAGCGGCGCCCCTCCTCCCAGCGGCGGCAGCCCAGCCGGCTCCCCAACCTCTACCGAGGAGGGCACCTCTGAGTCCGCCACCCCCGAGAGCGGCCCTGGCACCTCCACCGAGCCCAGCGAGGGCAGCGCACCCGGCAGCCCTGCCGGCAGCCCCACCTCCACAGAGGAGGGAACCAGCACCGAGCCCAGCGAAGGCAGCGCCCCAGGCACCAGCACCGAGCCTAGTGAG 3106 GGCGGCCCAAGCAGCGGCGCCCCUCCUCCCAGCGGCGGCAGCCCAGCCGGCUCCCCAACCUCUACCGAGGAGGGCACCUCUGAGUCCGCCACCCCCGAGAGCGGCCCUGGCACCUCCACCGAGCCCAGCGAGGGCAGCGCACCCGGCAGCCCUGCCGGCAGCCCCACCUCCACAGAGGAGGGAACCAGCACCGAGCCCAGCGAAGGCAGCGCCCCAGGCACCAGCACCGAGCCUAGUGAG 3121 dCasX491 See the sequence in Table 12 3049 CAGGAGAUUAAACGGAUCAACAAGAUCAGAAGAAGACUUGUGAAAGACAGCAACACCAAGAAGGCCGGCAAGACAGGCCCCAUGAAAACCCUGCUGGUUAGAGUGAUGACACCCGAUCUGAGAGAGCGGCUGGAAAACCUGAGAAAGAAGCCUGAAAAUAUCCCCCAGCCCAUCAGCAAUACA UCUAGAGCCAACCUGAAUAAGCUGCUGACCGAUUACACCGAAAUGAAGAAGGCGAUCCUGCAUGUGUACUGGGAAGAGUUCCAGAAGGACCCUGUGGGCCUGAUGAGCCGGGUGGCCCAGCCUGCCAGCAAGAAGAUCGAUCAGAACAAGCUGAAACCUGAGAUGGACGAGAAGGGCAACCUG ACCACCGCCGGCUUUGCCUGCUCUCAGUGUGGCCAGCCCCUGUUCGUGUACAAGCUGGAGCAGGUGUCUGAGAAGGGCAAGGCUUACACCAACUACUUCGGACGGUGCAAUGUGGCCGAGCACGAAAAGCUGAUCCUGCUGGCCCAGCUGAAGCCCGAGAAGGAUAGCGACGAAGCCGUGACA UAUAGCCUGGGAAAGUUUGGGCAGAGGGCCCUGGAUUUCUACAGCAUUCAUGUGACCAGGAGUCCACCCACCCGUGAAGCCCCUGGCCCAGAUCGCCGGAAACAGAUACGCCUCCGGACCUGUGGGAAAGGCCCUGAGCGACGCAUGUAUGGGCACAAUCGCCUCCUUCCUGUCUAAGUAC CAGGACAUCAUCAUCGAACACCAGAAGGUGGUGAAGGGCAACCAGAAGAGACUGGAGAGCCUGCGGGAGCUGGCCGGCAAGGAAAACCUGGAAUACCCUAGCGUGACCCUGCCACCUCAGCCUCACACCAAGGAGGGCGUUGAUGCCUACAACGAAGUGAUCGCCCGGGUGCGAAUGUGGGG AACCUGAACCUGUGGCAGAAGCUGAAGCUAAGCAGAGAUGAUGCCAAGCCUGCUGAGACUGAAGGGAUUCCCUUCCUUUCCUCUGGUCGAGACAGGCCAACGAAGUGGACUGGUGGGACAUGGUGUGUAACGUGAAGAAGCUGAUCAACGAGAAAAAGGAGGAUGGCAAGGUGUUUUGG CAGAAUCUGGCUGGCUACAAGAGACAGGAAGCCCUGAGACCAUACCUGAGCAGCGAGGAAGAUCGGAAGAAGGGAAAGAAAUUCGCUCGGUACCAGCUGGGCGACCUGCUGCUGCACCUGGAAAAGAAGCACGGCGAGGACUGGGGAAAGGUGUACGACGAGGCCUGGGAGCGGAUUGACAAG AAAGUGGAAGGCCUGAGCAAGCACAUCAAGCUGGAAGAGGAACGGAGAAGCGAGGACGCCCAGAGCAAGGCCGCCCUGACCGACUGGCUGCGGGCUAAGGCCAGCUUCGUGAUCGAGGGCCUGAAGGAGGCCGACAAGGACGAGUUCUGCAGAUGCGAGCUGAAGCUGCAGAAGUGGUACGGGG ACCUGCGGGGAAAGCCCUUCGCCAUCGAAGCCGAGAACAGCAUCCUGGACAUCAGCGGCUUCAGCAAGCAGUACAACUGUGCCUUCAUCUGGCAGAAGGACGGCGUGAAGAAGCUGAACCUGUACCUGAUCAUCAACUACUUCAAGGGCGGCAAGCUGCGGUUCAAGAAGAUCAAACCUGAAG CCUUCGAAGCCAACAGAUUCUACACCGUGAUCAACAAAAAGAGCGGCGAGAUCGUGCCCAUGGAGGUGAACUUCAACUUCGACGACCCCAACCUGAUCAUCCUGCCUGGCCUUUGGCAAGAGACAGGGCAGAGAAUUCAUCUGGAACGACCUGCUGUCCCUGGAAACCGGCAGCCUGAAGC UGGCCAACGGAAGAGUGAUCGAGAAGACACUGUACAACAGAAGAACCCGGCAGGAUGAGCCUGCCUGUUCGUGGCCCUGACCUUCGAGCGGCGGGAGGUCCUGGACUCCUCCAAUAUCAAACCAAUGAACCUGAUCGGCGUGGCAAGAGGCGAAAACAUCCCCGCCGUGAUCGCCCUGACCG ACCCCGAGGGCUGCCCACUGAGCCGGUUUAAGGAUAGCCUGGGAAACCCAACCCACAUCCUGAGAAUCGGCGAGAGCUAUAAGGAGAAGCAGCGGACCAUCCAGGCCAAGAAGGAGGUGGAGCAGCGGAGAGCCGGCGGCUACAGCCGGAAGUACGCCAGCAAAGCCAAGAAUCUGGCAGACGA UAUGGUGAGAAACACCGCUAGAGAUCUGCUGUACUACGCCGUGACCCAGGAUGCCAUGCUGAUCUUCGCCAACCUGAGCCGGGGCUUCGGCCGGCAGGGCAAGCGGACCUUCAUGGCCGAGAGACAGUACACACGGAUGGAGGACUGGCUGACCGCCAAGCUGGCCUACGAGGGCCUGAGCAA GACCUACCUGUCCAAGACACUGGCCCAGUACACCUCCAAGACAUGCAGCAACUGUGGGUUUACCAUCACCAGCGCCGACUACGACAGGGUGCUGGAGAAGCUGAAGAAGACAGCACAGGCUGGAUGACCACAAUUAACGGCAAGGAGCUGAAGGUGGAGGGCCAGAUUACCUACUACAACAG AUACAAGAGACAGAACGUAGUCAAGGACCUGUCCGUCGAGCUGGAUAGACUGAGCGAAGAAUCUGUGAACAACGACAUCUCCUCCUGGACAAAGGGCAGAAGCGGAGAAGCUCUGAGCCUCCUGAAGAAAAGAUUCUCCCAUAGACCCGUGCAGGAGAAGUUCGUGUGCCUGAACUGCGGCUU CGAGACACACGCAGCCGAGCAAGCCGCCCUGAACAUCGCCAGAUCCUGGCUGUUCCUGCGGAGCCAGGAGUACAAGAAAUACCAGACAAACAAGACAACCGGCAACACCGAUAAGAGAGCCUUCGUCGAGACCUGGCAGUCCUUUUACCGGAAGAAGCUUAAGGAGGUGUGGAAACCUGCCGUG 3122 Buffer + Connector CGGTCTGGCGGATCTGGCGGAGGCTCCACCAGC 3107 CGGUCUGGCGGAUCUGGCGGAGGCUCCACCAGC 3123 NLS + STOP codon + buffer sequence CCCAAGAAAAAGAGAAAAGTCTAATAGATAA 3108 CCCAAGAAAAAGAGAAAAGUCUAAUAGAUAA 3124 3'UTR GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG 3055 GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG 3125 Buffer sequence TCTAG 3056 UCUAG 3126 Poly(A) tail AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3109 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3109 LTRP5-ADD-ZIM3 5'UTR See above sequence 3047 See above sequence 3115 START codon + NLS + linker See above sequence 3104 See above sequence 3116 START codon + DNMT3A ADD domain ATGGAACGCCTCGTCTACGAGGTGCGGCAGAAGTGCAGAAACATCGAGGACATCTGCATCTCCTGCGGATCTCTGAACGTGACCCTGGAGCACCCACTGTTCATCGGCGGCATGTGCCAGAACTGTAAAAACTGTTTTCTGGAGTGTGCCTATCAATACGACGATGACGGCTACCAGAGCTACTGCACCATCTGTTGCGGCGGA AGAGAGGTGCTGATGTGTGGAAATAACAACTGCTGCCGGTGCTTCTGCGTGGAATGCGTGGACCTGCTGGTGGGCCCCGGCGCCGCCCAGGCCGCTATTAAGGAAGATCCTTGGAACTGCTACATGTGCGGCCACAAGGGCACATACGGCCTGCTGAGACGGAGAGAGGACTGGCCTAGCAGACTGCAGATGTTCTTCGCCAAT 3111 AUGGAACGCCUCGUACGAGGUGCGGCAGAAGUGCAGAAACAUCGAGGACAUCUGCAUCCUGCGGAUCUCUGAACGUGACCCUGGAGCACCCACUGUUCAUCGGCGGCAUGUGCCAGAACUGUAAAAACUGUUUUCUGGAGUGUGCCUAUCAAUACGACGAUGACGGCUACCAGAGCUACUGCACCAUCUGUUGCGGCGGA AGAGAGGUGCUGAUGUGUGGAAAUAACAACUGCUGCCGGUGCUUCUGCGUGGAAUGCGUGGACCUGCUGGUGGGCCCCGGCGCCGCCCAGGCCGCUAUUAAGGAAGAUCCUUGGAACUGCUACAUGUGCGGCCACAAGGGCACAUACGGCCUGCUGAGACGGAGAGAGGACUGGCCUAGCAGACUGCAGAUGUUCUUCGCCAAU 3127 DNMT3A catalytic domain AACCACGACCAGGAGTTCGACCCCCCTAAGGTGTACCCTCCCGTCCCCGCCGAGAAGAGAAAGCCCATCCGGGTCCTGAGCCTGTTCGATGGCATCGCCACCGGTCTGCTGGTGCTGAAGGACCTGGGCATCCAGGTGGATAGGTACATTGCCTCCGAGGTGTGCGAGGACTCCATCACCGTGGGAATGGTGCGTCATCAGGGCAAGATCATGTACGTGGGCGAC GTGCGGAGCGTGACACAGAAGCATATCCAGGAGTGGGGCCCTTTCGACCTGGTGATCGGCGGCAGCCCTTGCAATGACCTGAGCATCGTGAACCCAGCCCGGAAGGGCCTGTACGAGGGAACCGGCAGACTGTTCTTCGAGTTTTACAGACTGCTGCACGACGCCCGGCCTAAGGAAGGCGACGACCGGCCCTTTCTTTTGGCTGTTCGAGAATGTGGTGGCCATGG GAGTCAGCGACAAGCGGGATATTAGCCGGTTCCTGGAGAGCAACCCCGTGATGATCGATGCCAAGGAAGTGAGCGCCGCCCACCGGGCCAGATACTTCTGGGGCAATCTGCCTGGCATGAACAGACCCCTGGCCAGCACCGTGAACGACAAGCTGGAGCTGCAGGAGTGCCTGGAGCACGGCCGGATCGCCAAGTTCAGCAAGGTGAGAACCATCACCACCCGAAG CAACAGCATCAAACAAGGCAAGGACCAGCACTTTCCTGTGTTCATGAACGAAGGAGGACATCCTGTGGTGTACCGAGATGGAGAGAGTGTTCGGGTTCCCAGTCCACTACACAGATGTCAGCAACATGTCTAGACTGGCCAGACAGAGACTGCTGGGAAGAAGCTGGTCCGTCCCTGTGATCAGACACCTGTTCGCCCCTCTGAAGGAGTACTTCGCCTGCGTG 3112 AACCACGACCAGGAGUUCGACCCCCUAAGGUGUACCCUCCCGUCCCCGCCGAGAAGAGAAAGCCCAUCCGGUCCUGAGCCUGUUCGAUGGCAUCGCCACCGGUCUGCUGGUGCUGAAGGACCUGGGCAUCCAGGUGGAUAGGUACAUUGCCUCCGAGGUGUGCGAGGACUCCAUCACCGUGGGAAUGGUGCGUCAUCAGGGCAAGAUCAUGUACGUGGGCGAC GUGCGGAGCUGACACAGAAGCAUAUCCAGGAGUGGGGCCUUUCGACCUGGUGAUCGGCGGCAGCCCUUGCAAUGACCUGAGCAUCGUGAACCCAGCCCGGAAGGGCCUGUACGAGGGAACCGGCAGACUGUUCUUCGAGUUUACAGACUGCUGCACGACGCCCGGCCUAAGGAAGGCGACGACCGGCCCUUCUUUUGGCUGUUCGAGAAUGUGGUGGCCAUGG GAGUCAGCGACAAGCGGGAUAUUAGCCGGUUCCUGGAGAGCAACCCCGUGAUGAUCGAUGCCAAGGAAGUGAGCGCCGCCCACCGGGCCAGAUACUUCUGGGGCAAUCUGCCUGGCAUGAACAGACCCCUGGCCAGCACCGUGAACGACAAGCUGGAGCUGCAGGAGUGCCUGGAGCACGGCCGGAUCGCCAAGUUCAGCAAGGUGAGAACCAUCACCACCCGAAG CAACAGCAUCAAACAAGGCAAGGACCAGCACUUUCCUGUGUUCAUGAACGAAGGAGGACAUCCUGUGGUGUACCGAGAUGGAGAGUGUUCGGGUUCCCAGUCCACUACACAGAUGUCAGCAACAUGUCUAGACUGGCCAGACAGAGACUGCUGGGAAGAAGCUGGUCCGUCCCCUGUGAUCAGACACCUGUUCGCCCCUCUGAAGGAGUACUUCGCCUGCGUG 3128 Connector See above sequence 3060 See above sequence 3118 DNMT3L interaction domain See above sequence 3061 See above sequence 3119 Connector See above sequence 3105 See above sequence 3105 ZIM3-KRAB See the sequence in Table 12 3051 See above sequence 3120 Connector See above sequence 3106 See above sequence 3121 dCasX491 See above sequence 3049 See above sequence 3122 Buffer + Connector See above sequence 3107 See above sequence 3123 NLS + STOP codon + buffer sequence See above sequence 3108 See above sequence 3124 3'UTR See above sequence 3055 See above sequence 3125 Buffer sequence See above sequence 3056 See above sequence 3126 Poly(A) tail See above sequence 3109 See above sequence 3109 * The components are listed in 5' to 3' order within the construct Table 16 : Full-length RNA sequences of LTRP5-ZIM3 and LTRP5-ZIM3-ADD mRNA molecules evaluated in this example . Modification ' mψ ' = N1- methyl - pseudouridine LTRP molecules RNA- seq SEQ ID NO LTRP5-ZIM3 AAAmψAAGAGAGAAAAGAAGAGmψAAGAAGAAAmψAmψAAGAGCCACCAmψGGCCCCmψAAGAAGAAGCGmψAAAGmψGAGCCGGAmψGAACCACGAC CAGGAGmψmψCGACCCCCCmψAAGGmψGmψACCCmψCCCGmψCCCCGCCGAGAAGAGAAAGCCCAmψCCGGGmψCCmψGAGCCmψGmψmψCGAmψGGC AmψCGCCACCGGmψCmψGCmψGGmψGCmψGAAGGACCmψGGGCAmψCCAGGmψGGAmψAGGmψACAmψmψGCCmψCCGAGGmψGmψGCGAGGACmψCC AmψCACCGmψGGGAAmψGGmψGCGmψCAmψCAGGGCAAGAmψCAmψGmψACGmψGGGCGACGmψGCGGAGCGmψGACACAGAAGCAmψAmψCCAGGAGm ψGGGGCCCmψmψmψCGACCmψGGmψGAmψCGGCGGCAGCCCmψmψGCAAmψGACCmψGAGCAmψCGmψGAACCCAGCCCGGAAGGGCCmψGmψACGAG GGAACCGGCAGACmψGmψmψCmψmψCGAGmψmψmψmψACAGACmψGCmψGCACGACGCCCGGCCmψAAGGAAGGCGACGACCGGCCCmψmψCmψmψmψ mψGGCmψGmψmψCGAGAAmψGmψGGmψGGCCAmψGGGAGmψCAGCGACAAGCGGGAmψAmψmψAGCCGGmψmψCCmψGGAGAGCAACCCCGmψGAmψG AmψCGAmψGCCAAGGAAGmψGAGCGCCGCCCACCGGGCCAGAmψACmψmψCmψGGGGCAAmψCmψGCCmψGGCAmψGAACAGAACCCmψGGCCAGCACC GmψGAACGACAAGCmψGGAGCmψGCAGGAGmψGCCmψGGAGCACGGCCGGAmψCGCCAAGmψmψCAGCAAGGmψGAGAACCAmψCACCACCCGAAGCA ACAGCAmψCAAACAAGGCAAGGACCAGCACmψmψmψCCmψGmψGmψmψCAmψGAACGAGAAGGAGGACAmψCCmψGmψGGmψGmψACCGAGAmψGGAG AGAGmψGmψmψCGGGmψmψCCCAGmψCCACmψACACAGAmψGmψCAGCAACAmψGmψCmψAGACmψGGCCAGACAGAGACmψGCmψGGGAAGAAGCmψ GGmψCCGmψCCCmψGmψGAmψCAGACACCmψGmψmψCGCCCCmψCmψGAAGGAGmψACmψmψCGCCmψGCGmψGAGCAGCGGCAACAGCAACGCCAACA GCCGGGGCCCCAGCmψmψCmψCmψAGCGGCCmψGGmψGCCACmψGmψCCCmψGAGAGGGAGCCACAmψGGGCCCCAmψGGAGAmψCmψACAAAACCGm ψGAGCGCCmψGGAAGCGGCAGCCmψGmψGCGCGmψGCmψGAGCCmψGmψmψmψCGGAAmψAmψCGAmψAAAGmψCCmψGAAAAGCCmψGGGAmψmψCC mψGGAGAGCGGCmψCmψGGCmψCCGGCGGmψGGCACCCmψGAAGmψACGmψGGAGGAmψGmψGACAAACGmψGGmψCAGACGGGAmψGmψGGAGAAGm ψGGGGGCCCCmψmψCGAmψCmψGGmψGmψACGGCAGCACCCAACCCmψGGGCAGCmψCmψmψGmψGACCGGmψGCCCmψGGCmψGGmψACAmψGmψmψm ψCAGmψmψCCACCGGAmψCCmψGCAGmψACGCCCmψGCCGAGACAGGAGmψCCCAGCGGCCAmψmψCmψmψmψmψGGAmψmψmψmψCAmψGGACAACm ψmψGCmψGCmψGACCGAGGAmψGACCAGGAAACmψACCACmψCGGmψmψCCmψGCAGACCGAAGCCGmψGACCCmψGCAGGACGmψGAGAGGCCGGGA CmψACCAGAACGCCAmψGCGGGmψGmψGGmψCCAACAmψCCCmψGGACmψGAAAAGCAAGCACGCACCmψCmψGACCCCmψAAAGAAGAGGAGmψACC mψGCAGGCCCAGGmψGCGGAGCAGAAGCAAGCmψGGACGCCCCmψAAGGmψGGAmψCmψGCmψGGmψGAAGAAmψmψGCCmψCCmψGCCCCmψGAGAGA GmψACmψmψCAAGmψAmψmψmψCAGCCAGAAmψAGmψCmψGCCCCmψGGGAGGCAGCGGCGGCGGCAmψGAACAACmψCCCAGGGCAGAGmψGACCmψ mψCGAGGACGmψGACCGmψGAAmψmψmψmψACACAGGGAGAGmψGGCAGAGACmψGAACCCCGAGCAGAGAAACCmψGmψACCGGGAmψGmψGAmψGC mψGGAAAACmψACAGCAAmψCmψGGmψGmψCCGmψGGGCCAGGGCGAGACCACAAAGCCmψGACGmψGAmψCCmψGCGmψCmψGGAGCAGGGCAAGGA ACCCmψGGCmψGGAGGAGGAGGAGGmψGCmψGGGAAGCGGACGGGCCGAGAAGAACGGCGACAmψCGGCGGACAGAmψCmψGGAAGCCmψAAGGACGmψ GAAAGAAAGCCmψGGGCGGCCCAAGCAGCGGCGCCCCmψCCmψCCCAGCGGCGGCAGCCCAGCCGGCmψCCCCAACCmψCmψACCGAGGAGGGCACCmψCmψGAGmψCCGCCACCCCCGAGAGCGGCCCmψGGCACCmψCCACCGAGCCCAGCGAGGGCAGCGCACCCGGCAGCCCmψGCCGGCAGCCCCACCmψC CACAGAGGAGGGAACCAGCACCGAGCCCAGCGAAGGCAGCGCCCCAGGCACCAGCACCGAGCCmψAGmψGAGCAGGAGAmψmψAAACGGAmψCAACAAGAmψCAGAAGAAGACmψmψGmψGAAAGACAGCAACACCAAGAAGGCCGGCAAGACAGGCCCCAmψGAAAACCCmψGCmψGGmψmψAGAGmψGAmψGACA CCCGAmψCmψGAGAGAGCGGCmψGGAAAACCmψGAGAAAGAAGCCmψGAAAmψAmψCCCCCAGCCCAmψCAGCAAmψACAmψCmψAGAGCCAACCmψ GAAmψAAGCmψGCmψGACCGAmψmψACACCGAAAmψGAAGAAGGCGamψCCmψGCAmψGmψGmψACmψGGGAAGAGmψmψCCAGAAGGACCCmψGmψG GGCCmψGAmψGAGCCGGGmψGGCCCAGCCmψGCCAGCAAGAAGAmψCGAmψCAGAACAAGCmψGAAACCmψGAGAmψGGACGAGAAGGGCAACCmψGA CCACCGCCGGCmψmψmψGCCmψGCmψCmψCAGmψGmψGGCCAGCCCCmψGmψmψCGmψGmψACAAGCmψGGAGCAGGmψGmψCmψGAGAAGGGCAAGGC mψmψACACCAACmψACmψmψCGGACGGmψGCAAmψGmψGGCCGAGCACGAAAAGCmψGAmψCCmψGCmψGGCCCAGCmψGAAGCCCGAGAAGGAmψAG CGACGAAGCCGmψGACAmψAmψAGCCmψGGGAAAGmψmψmψGGGCAGAGGGCCCmψGGAmψmψmψCmψACAGCAmψmψCAmψGmψGACCAAGGAGmψC CACCCACCCCGmψGAAGCCCmψGGCCCAGAmψCGCCGGAAACAGAmψACGCCmψCCGGACCmψGmψGGGAAAGGCCCmψGAGCGACGCAmψGmψAmψ GGGCACAAmψCGCCmψCCmψmψCCmψGmψCmψAAGmψACCAGGACAmψCAmψCAmψCGAACACCAGAAGGmψGGmψGAAGGGCAACCAGAAGAGACmψG GAGAGCCmψGCGGGAGCmψGGCCGGCAAGGAAAACCmψGGAAmψACCCmψAGCGmψGACCCmψGCCACCmψCAGCCmψCACACCAAGGAGGGCGmψmψ GAmψGCCmψACAACGAAGmψGAmψCGCCCGGGmψGCGAAmψGmψGGGmψGAACCmψGAACCmψGmψGGCAGAAGCmψGAAGCmψAAGCAGAGAmψGAm ψGCCAAGCCmψCmψGCmψGAGACmψGAAGGGAmψmψCCCmψmψCCmψmψmψCCmψCmψGGmψCGAGAGACAGGCCAACGAAGmψGGACmψGGmψGGGA CAmψGGmψGmψGmψAACGmψGAAGAAGCmψGAmψCAACGAGAAAAAGGAGGAmψGGCAAGGmψGmψmψmψmψGGCAGAAmψCmψGGCmψGGCmψACAAG AGACAGGAAGCCCmψGAGACCAmψACCmψGAGCAGCGAGGAAGAmψCGGAAGAAGGGAAAGAAAmψmψCGCmψCGGmψACCAGCmψGGGCGACCmψGCmψGCmψGCACCmψGGAAAAGAAGCACGGCGAGGACmψGGGGAAAGGmψGmψACGACGAGGCCmψGGGAGCGGAmψmψGACAAGAAAGmψGGAAGGCCm ψGAGCAAGCACAmψCAAGCmψGGAAGAGGAACGGAGAAGCGAGGACGCCCAGAGCAAGGCCGCCCmψGACCGACmψGGCmψGCGGGCmψAAGGCCAGC mψmψCGmψGAmψCGAGGGCCmψGAAGGAGGCCGACAAGGACGAGmψmψCmψGCAGAmψGCGAGCmψGAAGCmψGCAGAAGmψGGmψACGGGGACCmψGC GGGGAAAGCCCmψmψCGCCAmψCGAAGCCGAGAACAGCAmψCCmψGGACAmψCAGCGGCmψmψCAGCAAGCAGmψACAACmψGmψGCCmψmψCAmψCm ψGGCAGAAGGACGGCGmψGAAGAAGCmψGAACCmψGmψACCmψGAmψCAmψCAACmψACmψmψCAAGGGCGGCAAGCmψGCGGmψmψCAAGAAGAmψC AAACCmψGAAGCCmψmψCGAAGCCAACAGAmψmψCmψACACCGmψGAmψCAACAAAAAGAGCGGCGAGAmψCGmψGCCCAmψGGAGGmψGAACmψmψC AACmψmψCGACGACCCCAACCmψGAmψCAmψCCmψGCCmψCmψGGCCmψmψmψGGCAAGAGACAGGGCAGAGAAmψmψCAmψCmψGGAACGACCmψGCm ψGmψCCCmψGGAAACCGGCAGCCmψGAAGCmψGGCCAACGGAAGAGmψGAmψCGAGAAGACACmψGmψACAACAGAAGAACCCGGCAGGAmψGAGCCm ψGCCCmψGmψmψCGmψGGCCCmψGACCmψmψCGAGCGGCGGGAGGmψCCmψGGACmψCCmψCCAAmψAmψCAAACCAAmψGAACCmψGAmψCGGCGmψ GGCAAGAGGCGAAAACAmψCCCCGCCmψGAmψCGCCCmψGACCGACCCCGAGGGCmψGCCCACmψGAGCCGGmψmψmψAAGGAmψAGCCmψGGGAAACCCAACCCACAmψCCmψGAGAAmψCGGCGAGAGCmψAmψAAGGAGAAGCAGCGGACCAmψCCAGGCCAAGAAGGAGGmψGGAGCAGCGGAGAGCCGGCG GCmψACAGCCGGAAGmψACGCCAGCAAAGCCAAGAAmψCmψGGCAGACGAmψAmψGGmψGAGAAACACCGCmψAGAGAmψCmψGCmψGmψACmψACGC CGmψGACCCAGGAmψGCCAmψGCmψGAmψCmψmψCGCCAACCmψGAGCCGGGGCmψmψCGGCCGGCAGGGCAAGCGGACCmψmψCAmψGGCCGAGAGA CAGmψACACACGGAmψGGAGGACmψGGCmψGACCGCCAAGCmψGGCCmψACGAGGGCCmψGAGCAAGACCmψACCmψGmψCCAAGACACmψGGCCCAG mψACACCmψCCAAGACAmψGCAGCAACmψGmψGGGmψmψmψACCAmψCACCAGCGCCGACmψACGACAGGGmψGCmψGGAGAAGCmψGAAGAAGACAGC AACAGGCmψGGAmψGACCACAAmψmψAACGGCAAGGAGCmψGAAGGmψGGAGGGCCAGAmψmψACCmψACmψACAACAGAmψACAAGAGACAGAACGm ψAGmψCAAGGACCmψGmψCCGmψCGAGCmψGGAmψAGACmψGAGCGAAGAAmψCmψGmψGAACAACGACAmψCmψCCmψCCmψGGACAAAGGGCAGAA GCGGAGAAGCmψCmψGAGCCmψCCmψGAAGAAAAGAmψmψCmψCCCAmψAGACCCGmψGCAGGAGAAGmψmψCGmψGmψGCCmψGAACmψGCGGCmψm ψCGAGACACACGCAGCCGAGCAAGCCGCCCmψGAACAmψCGCCAGAmψCCmψGGCmψGmψmψCCmψGCGGAGCCAGGAGmψACAAGAAAmψACCAGACA AACAAGACAACCGGCAACACCGAmψAAGAGAGCCmψmψCGmψCGAGACCmψGGCAGmψCCmψmψmψmψACCGGAAGAAGCmψmψAAGGAGGmψGmψGG AAACCmψGCCGmψGCGGmψCmψGGCGGAmψCmψGGCGGAGGCmψCCACCAGCCCCAAGAAAAAGAGAAAAGmψCmψAAmψAGAmψAAGCmψGCCmψmψC mψGCGGGGCmψmψGCCmψmψCmψGGCCAmψGCCCmψmψCmψmψCmψCmψCCCmψmψGCACCmψGmψACCmψCmψmψGGmψCmψmψmψGAAmψAAAGCCmψGAGmψAGGAAGmψCmψAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3129 LTRP5-ADD-ZIM3 AAAmψAAGAGAGAAAAGAAGAGmψAAGAAGAAAmψAmψAAGAGCCACCAmψGGCCCCmψAAGAAGAAGCGmψAAAGmψGAGCCGGAmψGGAACGCCmψCGmψCmψ ACGAGGmψGCGGCAGAAGmψGCAGAAACAmψCGAGGACAmψCmψGCAmψCmψCCmψGCGGAmψCmψCmψGAACGmψGACCCmψGGAGCACCCACmψGmψmψCAmψC GGCGGCAmψGmψGCCAGAACmψGmψAAAAACmψGmψmψmψmψCmψGGAGmψGmψGCCmψAmψCAAmψACGACGAmψGACGGCmψACCAGAGCmψACmψGCACCAmψ CmψGmψmψGCGGCGGAAGAGAGGmψGCmψGAmψGmψGmψGGAAAmψAACAACmψGCmψGCCGGmψGCmψmψCmψGCGmψGGAAmψGCGmψGGACCmψGCmψGGmψG GGCCCCGGCGCCGCCCAGGCCGCmψAmψmψAAGGAAGAmψCCmψmψGGAACmψGCmψACAmψGmψGCGGCCACAAGGGCACAmψACGGCCmψGCmψGAGACGGAGA GAGGACmψGGCCmψAGCAGACmψGCAGAmψGmψmψCmψmψCGCCAAmψAACCACGACCAGGAGmψmψCGACCCCCCmψAAGGmψGmψACCCmψCCCGmψCCCCGCC GAGAAGAGAAAGCCCAmψCCGGGmψCCmψGAGCCmψGmψmψCGAmψGGCAmψCGCCACCGGmψCmψGCmψGGmψGCmψGAAGGACCmψGGGCAmψCCAGGmψGGAm ψAGGmψACAmψmψGCCmψCCGAGGmψGmψGCGAGGACmψCCAmψCACCGmψGGGAAmψGGmψGCGmψCAmψCAGGGCAAGAmψCAmψGmψACGmψGGGCGACGmψG CGGAGCGmψGACACAGAAGCAmψAmψCCAGGAGmψGGGGCCCmψmψmψCGACCmψGGmψGAmψCGGCGGCAGCCCmψmψGCAAmψGACCmψGAGCAmψCGmψGAA CCCAGCCCGGAAGGGCCmψGmψACGAGGGAACCGGCAGACmψGmψmψCmψmψCGAGmψmψmψmψACAGACmψGCmψGCACGACGCCCGGCCmψAAGGAAGGCGACG ACCGGCCCmψmψCmψmψmψmψGGCmψGmψmψCGAGAAmψGmψGGmψGGCCAmψGGGAGmψCAGCGACAAGCGGGAmψAmψmψAGCCGGmψmψCCmψGGAGAGCAAC CCCGmψGAmψGAmψCGAmψGCCAAGGAAGmψGAGCGCCGCCCACCGGGCCAGAmψACmψmψCmψGGGGCAAmψCmψGCCmψGGCAmψGAACAGACCCCmψGGCCAG CACCGmψGAACGACAAGCmψGGAGCmψGCAGGAGmψGCCmψGGAGCACGGCCGGAmψCGCCAAGmψmψCAGCAAGGmψGAGAACCAmψCACCACCCGAAGCAACAG CAmψCAAACAAGGCAAGGACCAGCACmψmψmψCCmψGmψGmψmψCAmψGAACGAGAAGGAGGACAmψCCmψGmψGGmψGmψACCGAGAmψGGAGAGAGmψGmψmψC GGGmψmψCCCAGmψCCACmψACACAGAmψGmψCAGCAACAmψGmψCmψAGACmψGGCCAGACAGAGACmψGCmψGGGAAGAAGCmψGGmψCCGmψCCCmψGmψGAm ψCAGACACCmψGmψmψCGCCCCmψCmψGAAGGAGmψACmψmψCGCCmψGCGmψGAGCAGCGGCAACAGCAACGCCAACAGCCGGGGCCCCAGCmψmψCmψCmψAGC GGCCmψGGmψGCCACmψGmψCCCmψGAGAGGGAGCCACAmψGGGCCCCAmψGGAGAmψCmψACAAAACCGmψGAGCGCCmψGGAAGCGGCAGCCmψGmψGCGCGm ψGCmψGAGCCmψGmψmψmψCGGAAmψAmψCGAmψAAAGmψCCmψGAAAAGCCmψGGGAmψmψCCmψGGAGAGCGGCmψCmψGGCmψCCGGCGGmψGGCACCCmψGA AGmψACGmψGGAGGAmψGmψGACAAACGmψGGmψCAGACGGGAmψGmψGGAGAAGmψGGGGCCCCmψmψCGAmψCmψGGmψGmψACGGCAGCACCCAACCCCmψGG GCAGCmψCmψmψGmψGACCGGmψGCCCmψGGCmψGGmψACAmψGmψmψmψCAGmψmψCCACCGGAmψCCmψGCAGmψACGCCCmψGCCGAGACAGGAGmψCCCAGC GGCCAmψmψCmψmψmψmψGGAmψmψmψmψCAmψGGACAACmψmψGCmψGCmψGACCGAGGAmψGACCAGGAAACmψACCACmψCGGmψmψCCmψGCAGACCGAAGC CGmψGACCCmψGCAGGACGmψGAGAGGCCGGGACmψACCAGAACGCCAmψGCGGGmψGmψGGmψCCAACAmψCCCmψGGACmψGAAAAGCAAGCACGCACCmψCmψ GACCCCmψAAAGAAGAGGAGmψACCmψGCAGGCCCAGGmψGCGGAGCAGAAGCAAGCmψGGACGCCCCmψAAGGmψGGAmψCmψGCmψGGmψGAAGAAmψmψGCCm ψCCmψGCCCCmψGAGAGAGmψACmψmψCAAGmψAmψmψmψCAGCCAGAAmψAGmψCmψGCCCCmψGGGAGGCAGCGGCGGCGGCAmψGAACAACmψCCCAGGGCAG AGmψGACCmψmψCGAGGACGmψGACCGmψGAAmψmψmψmψACACAGGGAGAGmψGGCAGAGACmψGAACCCCGAGCAGAGAAACCmψGmψACCGGGAmψGmψGAm ψGCmψGGAAAACmψACAGCAAmψCmψGGmψGmψCCGmψGGGCCAGGGCGAGACCACAAAGCCmψGACGmψGAmψCCmψGCGmψCmψGGAGCAGGGCAAGGAACCCm ψGGCmψGGAGGAGGAGGAGGmψGCmψGGGAAGCGGACGGGCCGAGAAGAACGGCGACAmψCGGCGGACAGAmψCmψGGAAGCCmψAAGGACGmψGAAAGAAAGCCm ψGGGCGGCCCAAGCAGCGGCGCCCCmψCCmψCCCAGCGGCGGCAGCCCAGCCGGCmψCCCCAACCmψCmψACCGAGGAGGGCACCmψCmψGAGmψCCGCCACCCCC GAGAGCGGCCCmψGGCACCmψCCACCGAGCCCAGCGAGGGCAGCGCACCCGGCAGCCCmψGCCGGCAGCCCCACCmψCCACAGAGGAGGGAACCAGCACCGAGCCCAGCGAAGGCAGCGCCCCAGGCACCAGCACCGAGCCmψAGmψGAGCAGGAGAmψmψAAACGGAmψCAACAAGAmψCAGAAGAAGACmψmψGmψGAAAGACAGCAACA CCAAGAAGGCCGGCAAGACAGGCCCCAmψGAAAACCCmψGCmψGGmψmψAGAGmψGAmψGACACCCGAmψCmψGAGAGAGCGGCmψGGAAAACCmψGAGAAAGAAG CCmψGAAAAmψAmψCCCCAGCCCAmψCAGCAAmψACAmψCmψAGAGCCAACCmψGAAmψAAGCmψGCmψGACCGAmψmψACACGAAAmψGAAGAAGGCGAmψCC mψGCAmψGmψGmψACmψGGGAAGAGmψmψCCAGAAGGACCCmψGmψGGGCCmψGAmψGAGCCGGGmψGGCCCAGCCmψGCCAGCAAGAAGAmψCGAmψCAGAACA AGCmψGAAACCmψGAGAmψGGACGAGAAGGGCAACCmψGACCACCGCCGGCmψmψmψGCCmψGCmψCmψCAGmψGmψGGCCAGCCCCmψGmψmψCGmψGmψACAAG CmψGGAGCAGGmψGmψCmψGAGAAGGGCAAGGCmψmψACACCAACmψACmψmψCGGACGGmψGCAAmψGmψGGCCGAGCACGAAAAGCmψGAmψCCmψGCmψGGCC CAGCmψGAAGCCCGAGAAGGAmψAGCGACGAAGCCGmψGACAmψAmψAGCCmψGGGAAAGmψmψmψGGGCAGAGGGCCCmψGGAmψmψmψCmψACAGCAmψmψCAm ψGmψGACCAAGGAGmψCCACCCACCCCGmψGAAGCCCCmψGGCCCAGAmψCGCCGGAAACAGAmψACGCCmψCCGGACCmψGmψGGGAAAGGCCCmψGAGCGACGC AmψGmψAmψGGGCACAAmψCGCCmψCCmψmψCCmψGmψCmψAAGmψACCAGGACAmψCAmψCAmψCGAACACCAGAAGGmψGGmψGAAGGGCAACCAGAAGAGACm ψGGAGAGCCmψGCGGGAGCmψGGCCGGCAAGGAAAACCmψGGAAmψACCCmψAGCGmψGACCCmψGCCACCmψCAGCCmψCACACCAAGGAGGGCGmψmψGAmψGC CmψACAACGAAGmψGAmψCGCCCGGGmψGCGAAmψGmψGGGmψGAACCmψGAACCmψGmψGGCAGAAGCmψGAAGCmψAAGCAGAGamψGAmψGCCAAGCCmψCmψ GCmψGAGACmψGAAGGGAmψmψCCCmψmψCCmψmψmψCCmψCmψGGmψCGAGAGACAGGCCAACGAAGmψGGACmψGGmψGGGACAmψGGmψGmψGmψAACGmψG AAGAAGCmψGAmψCAACGAGAAAAAGGAGGAmψGGCAAGGmψGmψmψmψmψGGCAGAAmψCmψGGCmψGGCmψACAAGAGACAGGAAGCCCmψGAGACCAmψACCm ψGAGCAGCGAGGAAGAmψCGGAAGAAGGGAAAGAAAmψmψCGCmψCGGmψACCAGCmψGGGCGACCmψGCmψGCmψGCACCmψGGAAAAGAAGCACGGCGAGGACm ψGGGGAAAGGmψGmψACGACGAGGCCmψGGGAGCGGAmψmψGACAAGAAAGmψGGAAGGCCmψGAGCAAGCACAmψCAAGCmψGGAAGAGGAACGGAGAAGCGAGG ACGCCCAGAGCAAGGCCGCCCmψGACCGACmψGGCmψGCGGGCmψAAGGCCAGCmψmψCGmψGAmψCGAGGGCCmψGAAGGAGGCCGACAAGGACGAGmψmψCmψG CAGAmψGCGAGCmψGAAGCmψGCAGAAGmψGGmψACGGGGACCmψGCGGGGAAAGCCCmψmψCGCCAmψCGAAGCCGAGAACAGCAmψCCmψGGACAmψCAGCGGC mψmψCAGCAAGCAGmψACAACmψGmψGCCmψmψCAmψCmψGGCAGAAGGACGGCGmψGAAGAAGCmψGAACCmψGmψACCmψGAmψCAmψCAACmψACmψmψCAAG GGCGGCAAGCmψGCGGmψmψCAAGAAGAmψCAAACCmψGAAGCCmψmψCGAAGCCAACAGAmψmψCmψACACCGmψGAmψCAACAAAAAGAGCGGCGAGAmψCGmψ GCCCAmψGGAGGmψGAACmψmψCAACmψmψCGACGACCCCAACCmψGAmψCAmψCCmψGCCmψCmψGGCCmψmψmψGGCAAGAGACAGGGCAGAGAAmψmψCAmψ CmψGGAACGACCmψGCmψGmψCCCmψGGAAACCGGCAGCCmψGAAGCmψGGCCAACGGAAGAGmψGAmψCGAGAAGACACmψGmψACAACAGAAGAACCCGGCAGG AmψGAGCCmψGCCCmψGmψmψCGmψGGCCCmψGACCmψmψCGAGCGGCGGGAGGmψCCmψGGACmψCCmψCCAAmψAmψCAAACCAAmψGAACCmψGAmψCGGCGm ψGGCAAGAGGCGAAAACAmψCCCCGCCgmψGAmψCGCCCmψGACCGACCCGAGGGCmψGCCCACmψGAGCCGGmψmψmψAAGGAmψAGCCmψGGGAAACCCAACC CACAmψCCmψGAGAAmψCGGCGAGAGCmψAmψAAGGAGAAGCAGCGGACCAmψCCAGGCCAAGAAGGAGGmψGGAGCAGCGGAGAGCCGGCGGCmψACAGCCGGAA GmψACGCCAGCAAAGCCAAGAAmψCmψGGCAGACGAmψAmψGGmψGAGAAACACCGCmψAGAGAmψCmψGCmψGmψACmψACGCCGmψGACCCAGGAmψGCCAmψG CmψGAmψCmψmψCGCCAACCmψGAGCCGGGGCmψmψCGGCCGGCAGGGCAAGCGGACCmψmψCAmψGGCCGAGAGACAGmψACACACGGAmψGGAGGACmψGGCmψ GACCGCCAAGCmψGGCCmψACGAGGCCmψGAGCAAGACCmψACCmψGmψCCAAGACACmψGGCCCAGmψACACCmψCCAAGACAmψGCAGCAACmψGmψGGGmψm ψmψACCAmψCACCAGCGCCGACmψACGACAGGGmψGCmψGGAGAAGCmψGAAGAAGACAGCAACAGGCmψGGAmψGACCACAAmψmψAACGGCAAGGAGCmψGAAG GmψGGAGGGCCAGAmψmψACCmψACmψACAACAGAmψACAAGAGACAGAACGmψAGmψCAAGGACCmψGmψCCGmψCGAGCmψGGAmψAGACmψGAGCGAAGAAmψ CmψGmψGAACAACGACAmψCmψCCmψCCmψGGACAAAGGGCAGAAGCGGAGAAGCmψCmψGAGCCmψCCmψGAAGAAAAGAmψmψCmψCCCAmψAGACCCGmψGCA GGAGAAGmψmψCGmψGmψGCCmψGAACmψGCGGCmψmψCGAGACACACGCAGCCGAGCAAGCCGCCCmψGAACAmψCGCCAGAmψCCmψGGCmψGmψmψCCmψGCG GAGCCAGGAGmψACAAGAAAmψACCAGACAAACAAGACAACCGGCAACACCGAmψAAGAGAGCCmψmψCGmψCGAGACCmψGGCAGmψCCmψmψmψmψACCGGAAG AAGCmψmψAAGGAGGmψGmψGGAAACCmψGCCGmψGCGGmψCmψGGCGGAmψCmψGGCGGAGGCmψCCACCAGCCCCAAGAAAAAGAGAAAAGmψCmψAAmψAGAm ψAAGCmψGCCmψmψCmψGCGGGGCmψmψGCCmψmψCmψGGCCAmψGCCCmψmψCmψmψCmψCmψCCCmψmψGCACCmψGmψACCmψCmψmψGGmψCmψmψmψGAAm ψAAAGCCmψGAGmψAGGAAGmψCmψAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3130 Table 17 : Activity and average CpG methylation percentage (mCpG) of the 16 spacers measured by ELISA and RT-qPCR Spacer ID ELISA reduction% RT-qPCR reduction% Average % mCpG TG-06-001 -54.60 -53.66 59.00 TG-06-133 -26.60 -58.51 42.90 TG-06-139 -51.50 -63.03 55.90 TG-06-141 -53.70 -58.85 53.70 TG-06-142 -18.50 -31.76 28.40 TG-06-143 -27.20 -51.60 46.10 TG-06-144 -28.10 -41.68 54.70 TG-06-146 -22.10 -45.31 31.00 TG-06-149 -57.50 -42.07 56.90 TG-06-150 -56.30 -55.68 56.40 TG-06-154 -66.50 -55.28 45.50 TG-06-157 -55.60 -52.86 54.40 TG-06-158 -56.50 -57.04 49.40 TG-06-160 -42.00 -54.91 57.30 TG-06-167 -37.80 -50.29 46.00 TG-06-168 -39.90 -50.34 48.80 initial N/A N/A 6.70 NT N/A N/A 1.50 Table 18 : Persistence and specificity of the previous 16 spacers determined by bisulfate sequencing and RNA-seq Spacer ID Δ% PCSK9 at Week 9 vs Week 1 Off-target differentially expressed genes# TG-06-154 -22.30 0 TG-06-157 -28.30 1 TG-06-001 -63.50 2 TG-06-141 -10.80 1 TG-06-149 -26.70 0 TG-06-158 0.40 376 TG-06-150 -35.50 2 TG-06-160 -23.50 18 TG-06-139 -34.90 twenty four TG-06-144 -21.30 0 TG-06-133 -29.40 6 Example 2 : Demonstration that mRNA encoding an LTRP containing an ADD domain can induce repression of endogenous loci in multiple human cell lines

Experiments were performed to demonstrate that mRNA encoding an LTRP containing an ADD domain can induce long-term repression of endogenous target loci in various human cell lines when delivered as mRNA co-transfected with a targeting gRNA. Materials and Methods mRNA Generation:

mRNA encoding the following molecules were generated by IVT following similar methods as described in Example 1: 1) catalytically active CasX 676, 2) dXR1, and 3) LTRP5-ADD-ZIM3. The sequences encoding these molecules were codon optimized using a codon usage table in addition to using publicly available codon optimization tools and adjusting parameters such as GC content. The DNA and mRNA sequences encoding catalytically active CasX 676 are shown in Tables 19 and 20, respectively. The DNA and mRNA sequences encoding dXR1 are shown in Tables 12 and 13, respectively. The DNA and mRNA sequences encoding LTRP5-ZIM3-ADD are shown in Tables 15 and 16, respectively. Table 19 : Coding sequences of catalytically active CasX 676 mRNA molecules evaluated in this example * CasX mRNA ID Component (ID) describe DNA Sequence SEQ ID NO: CasX 676 mRNA 5'UTR TriLink AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC 3047 START codon + c-MYC NLS ATGGCCCCTGCTGCCAAGAGAGTGAAGCTGGATAGCAGA 3133 CasX 676 CAGGAGATCAAGCGGATTAATAAAATTCGGAGAAGACTGGTGAAGGATTCTAACACAAAGAAGGCTGGCAAGACACGGGGCCCTATGAAGACACTGCTGGTGAGAGTGATGACACCCGACCTGAGAGAAAGACTGGAAAACCTGAGAAAGAAGCCTGAGAATATCCCCCAGCCCATCAGCAAC ACAAGCCGGGCCAACCTGAATAAGCTGCTGACCGACTACACCGAAATGAAGAAGGCCATCCTGCAGTGTATTGGGAAGAGTTCCAGAAAGACCCAGTCGGCCTGATGAGCAGAGTGGCTCAGCCTGCCAGCAAGAAGATCGATCAGAACAAGCTGAAGCCCGAAATGGACGAGAAGGGGAACC TGACAACCGCCGGCTTTGCCTGTAGCCAGTGCGGCCAGCCCCTGTTTGTGTACAAACTGGAACAGGTGAGCGAAAAGGGCAAGGCTTACACGAATTACTTCGGCAGATGCAACGTGGCCGAGCACGAGCTGATCAAGCTGGCCCAGCTGAAGCCTGAGAAGGATAGCGATGAGGCAGTGA CATATTCCCTGGGCAAGTTCGGACAGCGGGCCCTGGATTTTTATTCCATTCATGTGACCAAGGAATCCACCCACCCCGTCAAGCCTCTTGCCCAAATTGCCGGCAACAGATACGCCTCCAGCCCCGTGGGCAAGGCCCTGAGCGACGCCTGTATGGGCACCATCGCCAGCTTCCTGTCTAAGTA CCAGGACATTCATCGAGCACCAGAAGGTGGTGAAGGGCAACCAGAAGAGACTGGAGAGCCTGCGCGAGCTGGCCGGCAAGGAAAACCTGGAGTATCCTAGCGTGACCCTGCCTCCTCAGCCTCATACAAAGGAGGGCGTGGATGCCTACAACGAAGTGATCGCCCGGGTGCGGATGTGGGT GAACCTGAATCTGTGGCAGAAGCTGAAGCTGTCTAGAGACGACGCCAAGCCCCTGCTGAGACTGAAGGGCTTCCCCAGCTTCCCTCTGGTGGAGAGACAGGCAAATGAAGTGGACTGGTGGGACATGGTGTGTAACGTGAAGAAGCTGATCAATGAGAAGAAGGAGGACGGCAAAGTGTTCTGG CAGAATCTGGCCGGCTACAAGCGTCAGGAGGCCCTGCGGCCCTAACCTGAGCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGTTCGCCCGGTATCAGCTGGGGGGACCTGCTGCTGCACCTCGAGAAGAAGCACGGCGAAGACTGGGGGAAGGTGTACGATGAGGCCTGGGAGCGGATCGATAAG AAGGGTGGAGGGCCTGAGCAAGCACATCAAGCTGGAGGAGGAACGGAGATCTGAGGACGCCCAGAGCAAGGCCGCCCTGACCGACTGGCTGAGAGCCAAGGCCAGCTTCGTCATCGAGGGGCTGAAGGAGGCCGACAAGGACGAGTTCTGCCGGTGCGAACTGAAGCTGCAGAAGTGGTACGGAG ATCTGAGAGGCAAACCTTTCGCCATCGAGGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCAGTACAACTGCGCCTTTATTTGGCAGAAGGACGGAGTGAAGAAGCTGAACCTGTACCTGATCATCAACTATTTCAAGGGCGGCAAGCTGAGATTCAAGAAGATCAAGCCTGAAG CCTTCGAGGCCACAGATTCTACACCGTGATTAACAAGAAAAGCGGAGAGATCGTGCCAATGGAAGTGAACTTCAACTTCGACGACCCTAACCTGATCATCCTGCCCCTGGCATTTGGCAAGCGGCAGGGCAGAGAGTTCATCTGGAACGACCTGCTGTCTCTGGAGACCGGCAGCCTGAAGCT GGCCAACGGCAGAGTGATCGAGAAGACACTGTACAACAGACGAACCAGACAAGACGAGCCCGCCCTGTTTGTGGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGCAATATCAAGCCTATGAACCTGATCGGCGTGGACCGGGGCGAGAACATCCCTGCCGTGATCGCCCTTACCGA CCCCGAGGGATGCCCTCTGAGCCGGTTTAAAGACAGCCTGGGCAACCCTACCCACATCCTGAGAATTGGCGAGTCCTACAAGGAGAAGCAGAGAACCATCCAGGCCAAGAAGGAGGTGGAGCAGCGGCGGGCTGGCGGCTACTCCCGGAAGTACGCCAGCAAGGCCAAGAACCTGGCCGACGAC ATGGTTAGAAATACCGCCAGAGACCTCCTGTACTACGCTGTGACCCAGGACGCCATGCTGATCTTCGAGAACCTGAGCAGAGGCTTCGGCAGACAGGGCAAGAGAACCTTCATGGCCGAGAGACAGTACACCCGGATGGAGGACTGGCTGACCGCCAAGCTGGCCTACGAGGGCCTGCCCTCT AAGACCTACCTGTCCAAGACCTTGGCACAGTACACCAGCAAGACATGCTCTAACTGCGGCTTCACAATCACGAGCGCCGACTACGACCGGGTGCTGGAGAAACTGAAGAAGACCGCCACAGGCTGGATGACCACCATTAACGGCAAGGAGCTGAAGGTGGAGGGCCAGATCACCTACTACAACA GGTACAAACGGCAGAACGTGGTGAAGGACCTGAGCGTGGAACTGGATAGACTGAGCGAGGAAAGCGTAAACAATGACATCAGCAGCTGGACCAAGGGCCGGAGCGGCGAGGCCCTGAGCCTGCTGAAGAAGAGATTCTCCCACAGACCAGTGCAGGAGAAGTTCGTGTGTCTGAACTGCGGCTT CGAGACCCACGCCGACGAGCAAGCCGCCCTGAACATCGCCCGGTCTTGGCTTTTCCTGCGGAGCCAGGAGTACAAGAAGTACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAGCCTTCGTCGAGACCTGGCAGAGCTTCTACAGAAAGAAGCTGAAGGAGGTGTGGAAGCCTGCCGTG 3134 c-MYC NLS + STOP codon GGAAGCCCCGCTGCCAAGAGAGTGAAGCTGGACTAATAGATAA 3135 3'UTR Mouse HBA GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG 3055 XbaI restriction site (partial) TCTAG 3056 Poly(A) tail AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3057 * The components are listed in 5' to 3' order within the construct Table 20 : Full-length RNA sequences of catalytically active CasX 676 mRNA molecules evaluated in this example . Modification ' mψ ' = N1- methyl - pseudouridine CasX mRNA RNA- seq SEQ ID NO: CasX 676 mRNA AAAmψAAGAGAGAAAAGAAGAGmψAAGAAGAAAmψAmψAAGAGCCACCAmψGGCCCCmψGCmψGCCAAGAGAGmψGAAGCmψGGAmψAGCAGACAGGAGamψCAAGCGGAmψmψAA mψAAAAmψmψCGGAGAAGACmψGGmψGAAGGAmψmψCmψAACACAAAGAAGGCmψGGCAAGACACGGGGCCCmψAmψGAAGACACmψGCmψGGmψGAGAGmψGAmψGACACCCGACC mψGAGAGAAAGACmψGGAAAACCmψGAGAAAGAAGCCmψGAGAAmψAmψCCCCCAGCCCAmψCAGCAACACAAGCCGGGCCAACCmψGAAmψAAGCmψGCmψGACCGACmψACACCG AAAmψGAAGAAGGCCAmψCCmψGCACGmψGmψAmψmψGGGAAGAGmψmψCCAGAAAGACCCAGmψCGGCCmψGAmψGAGCAGAGmψGGCmψCAGCCmψGCCAGCAAGAAGAmψCGAm ψCAGAACAAGCmψGAAGCCCGAAAmψGGACGAGAAGGGGAACCmψGACAACCGCCGGCmψmψmψGCCmψGmψAGCCAGmψGCGGCCAGCCCCmψGmψmψmψGmψGmψACAAACmψGG AACAGGmψGAGCGAAAAGGGCAAGGCmψmψACACGAAmψmψACmψmψCGGCAGAmψGCAACGmψGGCCGAGCACGAGAAGCmψGAmψCAAGCmψGGCCCAGCmψGAAGCCmψGAGAA GGAmψAGCGAmψGAGGCAGmψGACAmψAmψmψCCCmψGGGCAAGmψmψCGGACAGCGGGCCCmψGGAmψmψmψmψmψAmψmψCCAmψmψCAmψGmψGACCAAGAAmψCCACCCACC CCGmψCAAGCCmψCmψmψGCCCAAAmψmψGCCGGCAACAGAmψACGCCmψCCAGCCCGmψGGGCAAGGCCCmψGAGCGACGCCmψGmψAmψGGGCACCAmψCGCCAGCmψmψCCmψ GmψCmψAAGmψACCAGGACAmψmψAmψCAmψCGAGCACCAGAAGGmψGGmψGAAGGGCAACCAGAAGAGACmψGGAGAGCCmψGCGCGAGCmψGGCCGGCAAGGAAAACCmψGGAGm ψAmψCCmψAGCGmψGACCCmψGCCmψCCmψCAGCCmψCAmψACAAAGGAGGGCGmψGGAmψGCCmψACAACGAAGmψGAmψCGCCCGGGmψGCGGAmψGmψGGGmψGAACCmψGAAm ψCmψGmψGGCAGAAGCmψGAAGCmψGmψCmψAGAGACGACGCCAAGCCCCmψGCmψGAGACmψGAAGGGCmψmψCCCCAGCmψmψCCCmψCmψGGmψGGAGAGACAGGCAAAmψGAA GmψGGACmψGGmψGGGACAmψGGmψGmψGmψAACGmψGAAGAAGCmψGAmψCAAmψGAGAAGAAGGAGGACGGCAAAGmψGmψmψCmψGGCAGAAmψCmψGGCCGGCmψACAAGCGm ψCAGGAGGCCCmψGCGGCCCmψACCmψGAGCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGmψmψCGCCCGGmψAmψCAGCmψGGGGGACCmψGCmψGCmψGCACCmψCGAGAAGAA GCACGGCGAAGACmψGGGGGAAGGmψGmψACGAmψGAGGCCmψGGGAGCGGAmψCGAmψAAGAAGGmψGGAGGGCCmψGAGCAAGCACAmψCAAGCmψGGAGGAGGAACGGAGAmψC mψGAGGACGCCCAGAGCAAGGCCGCCCmψGACCGACmψGGCmψGAGAGCCAAGGCCAGCmψmψCGmψCAmψCGAGGGGCmψGAAGGAGGCCGACAAGGACGAGmψmψCmψGCCGGmψ GCGAACmψGAAGCmψGCAGAAGmψGGmψACGGAGAmψCmψGAGAGGCAAACCmψmψmψCGCCAmψCGAGGCCGAGAACAGCAmψCCmψGGACAmψCAGCGGCmψmψCAGCAAGCAGm ψACAACmψGCGCCmψmψmψAmψmψmψGGCAGAAGGACGGAGmψGAAGAAGCmψGAACCmψGmψACCmψGAmψCAmψCAACmψAmψmψmψCAAGGGCGGCAAGCmψGAGAmψmψCAAG AAGAmψCAAGCCmψGAAGCCmψmψCGAGGCCAACAGAmψmψCmψACACCGmψGAmψmψAACAAGAAAAGCGGAGAGAmψCGmψGCCAAmψGGAAGmψGAACmψmψCAACmψmψCGAC GACCCmψAACCmψGAmψCAmψCCmψGCCCCmψGGCAmψmψmψGGCAAGCGGCAGGGCAGAGAGmψmψCAmψCmψGGAACGACCmψGCmψGmψCmψCmψGGAGACCGGCAGCCmψGAA GCmψGGCCAACGGCAGAGmψGAmψCGAGAAGACACmψGmψACAACAGACGAACCAGACAAGACGAGCCCGCCCmψGmψmψmψGmψGGCCCmψGACCmψmψCGAGAGAAGAGAGGmψG CmψGGACAGCAGCAAmψAmψCAAGCCmψAmψGAACCmψGAmψCGGCGmψGGACCGGGGCGAGAACAmψCCCmψGCCGmψGAmψCGCCCmψmψACCGACCCCGAGGGAmψGCCCmψCm ψGAGCCGGmψmψmψAAAGACAGCCmψGGGCAACCmψACCCACAmψCCmψGAGAAmψmψGGCGAGmψCCmψACAAGGAGAAGCAGAGAACCAmψCCAGGCCAAGAAGGAGGmψGGAG CAGCCGGCGGGCmψGGCGGCmψACmψCCCGGAAGmψACGCCAGCAAGGCCAAGAACCmψGGCCGACGACAmψGGmψmψAGAAAmψACCGCCAGAGACCmψCCmψGmψACmψACGCmψG mψGACCCAGGACGCCAmψGCmψGAmψCmψmψCGAGAACCmψGAGCAGAGGCmψmψCGGCAGACAGGGCAAGAGAACCmψmψCAmψGGCCGAGAGACAGmψACACCCGGAmψGGAGGA CmψGGCmψGACCGCCAAGCmψGGCCmψACGAGGGCCmψGCCCmψCmψAAGACCmψACCmψGmψCCAAGACCmψmψGGCACAGmψACACCAGCAAGACAmψGCmψCmψAACmψGCGGC mψmψCACAAmψCACGAGCGCCGACmψACGACCGGGmψGCmψGGAGAAACmψGAAGAAGACCGCCACAGGCmψGGAmψGACCACCAmψmψAACGGCAAGGAGCmψGAAGGmψGGAGGG CCAGAmψCACCmψACmψACAACAGGmψACAAACGGCAGAACGmψGGmψGAAGGACCmψGAGCGmψGGAACmψGGAmψAGACmψGAGCGAGGAAAGCGmψAAACAAmψGACAmψCAGC AGCmψGGACCAAGGGCCGGAGCGGCGAGGCCCmψGAGCCmψGCmψGAAGAAGAGAmψmψCmψCCCACAGACCAGmψGCAGGAGAAGmψmψCGmψGmψGmψCmψGAACmψGCGGCmψm ψCGAGACCCACGCCGACGAGCAAGCCGCCCmψGAACAmψCGCCCGGmψCmψmψGGCmψmψmψmψCCmψGCGGAGCCAGGAGmψACAAGAAGmψACCAGACAAACAAGACCACAGGCA ACACAGACAAGAGAGCCmψmψCGmψCGAGACCmψGGCAGAGCmψmψCmψACAGAAAGAAGCmψGAAGGAGGmψGmψGGAAGCCmψGCCGmψGGGAAGCCCCGCmψGCCAAGAGAGmψ GAAGCmψGGACmψAAmψAGAmψAAGCmψGCCmψmψCmψGCGGGGGCmψmψGCCmψmψCmψGGCCAmψGCCCmψmψCmψmψCmψCmψCCCmψmψGCACCmψGmψACCmψCmψmψGGmψC mψmψmψGAAmψAAAGCCmψGAGmψAGGAAGmψCmψAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3136 Synthesis of gRNA:

The gRNA targeting the human PCSK9 locus was designed using gRNA scaffold 316 and chemically synthesized. In addition, B2M targeting gRNA was used as an experimental control. The sequences of the targeting spacers evaluated in this example are listed in Table 21. Table 21 : Sequences of the spacers evaluated in this example Spacer ID Target Targeting spacer sequence (RNA) SEQ ID NO 7.37 Human B2M GGCCGAGAUGUCUCGCUCCG 3137 6.1 Human PCSK9 GAGGAGGACGGCCUGGCCGA 1834 6.125 Human PCSK9 AGAAAGAGCAAGCCUCAUGU 1845 6.138 Human PCSK9 AGGGAUUUAUACUACAAAGA 1852 6.153 Human PCSK9 GUUAAUGUUUAAUCAGAUAG 1866 6.157 Human PCSK9 GGGUCUGAGCCUGGAGGAGU 1869 6.172 Human PCSK9 GCUGAAACAGAUGGAAUACU 1879 6.181 Human PCSK9 UCAUCUGCACUCGUGGCCAC 2228 mRNA and gRNA were transfected into HepG2 cells, Hep3B cells, and Huh7 cells, and ELISA was performed to assess PCSK9 secretion:

The following three human hepatocellular carcinoma cell lines were used in this experiment: HepG2 cells, Hep3B cells, and Huh7 cells. Approximately 15,000 cells of each cell line were seeded per well; the next day, the seeded cells were transfected with mRNA encoding catalytically active CasX 676, dXR1, or LTRP5-ADD-ZIM3 and gRNA with scaffold 316 and a spacer targeting the B2M or PCSK9 locus (see Table 21 for specific spacers and sequences). The culture supernatant was collected 4 days after transfection to assess PCSK9 secretion levels by ELISA, and the PCSK9 secretion levels were normalized to total cell counts and are shown in Figures 3 to 5. Treated Huh7 cells were continued to be cultured and culture supernatants were collected 14 and 27 days after transfection for measurement of PCSK9 secretion by ELISA. As an additional experimental control, PCSK9 secretion was also measured in culture supernatants collected from wells containing untreated naive cells. Results:

HepG2 cells, Hep3B cells, and Huh7 cells were transfected with mRNA encoding catalytically active CasX 676, dXR1, or LTRP5-ADD-ZIM3 and gRNA targeting B2M or PCSK9 loci, and secreted PCSK9 levels were measured. Quantification of normalized PCSK9 secretion levels for each condition 4 days after transfection is depicted in Figures 3 to 5. The data show that the most efficient attenuation of PCSK9 secretion by CasX 676, dXR1, or LTRP5-ADD-ZIM3 was observed in Huh7 cells, while HepG2 cells did not show effective attenuation of secreted PCSK9 levels (Figures 3 to 5). Meanwhile, Hep3B cells showed overall low PCSK9 secretion levels, indicating that among the cell lines used, the Hep3B cell line was the least suitable for inducing and exhibiting PCSK9 inhibition (Figures 3 to 5).

After transfection, treated Huh7 cells were cultured until day 27, and PCSK9 secretion was measured at day 14 and day 27. The bar graph in FIG. 6 shows the quantitative results of PCSK9 inhibition at day 4, day 14, and day 27 time points, which are shown as PCSK9 gene attenuation relative to the level of the initial control detected at the day 4 time point. The data show that treatment of Huh7 cells with LTRP5-ADD-ZIM3 with gRNAs having spacers 6.138 and 6.157 caused the most effective inhibition of PCSK9 secretion, and this inhibition lasted until day 27 after transfection ( FIG. 6 ). Similarly, persistent gene attenuation was observed when Huh7 cells were treated with catalytically active CasX 676 and spacer 6.1. Although treatment with dXR1 and spacer 6.138 caused an initial strong inhibition at day 4, this inhibitory effect was transient, as PCSK9 secretion levels returned to baseline levels at days 14 and 27 post-transfection (Figure 6). As expected, treatment with any of the three mRNA molecules and spacer 7.37 targeting the B2M locus did not affect PCSK9 secretion levels during this time course experiment.

These experiments demonstrate that the use of LTRP molecules with ADD domains and appropriate targeting spacers can induce long-term silencing of endogenous target loci in various human cell lines. These findings also suggest that LTRP molecules with ADD domains can be co-delivered to cells as mRNAs with targeting gRNAs to induce repression. Example 3 : Design of modified gRNAs and their evaluation in improving editing when delivered with CasX mRNA in vitro and in vivo

Experiments were performed to identify novel gRNA variant sequences and to demonstrate that chemical modification of these gRNA variants enhances the editing efficiency of the CasX:gRNA system when delivered with CasX mRNA in vitro. Materials and Methods: Synthesis of gRNA:

All gRNAs tested in this example were chemically synthesized and derived from gRNA scaffolds 174, 235, and 316. The sequences of gRNA scaffolds 174, 235, and 316 and their chemical modification profiles are listed in Table 22. The sequences of the resulting gRNAs (including spacers targeting PCSK9 , B2M , or ROSA26 ) and their chemical modification profiles analyzed in this example are listed in Table 23. Schematic diagrams of the structures of gRNA scaffold variants 174, 235, and 316 are shown in Figures 7 to 9, respectively, and the sites of chemical modification of the gRNA variants are schematically shown in Figures 10, 11, 12, 13, and 14. Table 22 : Overview of gRNA scaffold sequences and their different chemical modifications ( indicated by model number ) , where " NNNNNNNNNNNNNNNNNNNN " is the reserved position for the spacer. Chemical modification : * = phosphorothioate bond ; m = 2'OMe modification gRNA scaffold ( type) gRNA sequences SEQ ID NO: 174 (v0) ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGNNNNNNNNNNNNNNNNNN 2947 174 (v1) mA*mC*mU*GGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGNNNNNNNNNNNNNNNNNmN*mN*mN 2948 174 (v2) mA*mC*mU*GGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUUCGGAGGGAGCAUCAAAGNNNNNNNNNNNNNNNNNN*mU*mU*mU 2949 174 (v3) mA*mC*mU*mGmGmCmGmCmUmUmUmUmAmUmCmUmGmAmUUACUUUGmAmGmAmGmCmCmAmUmCmAmCmCAGCGAmCmUAUmGmUmCmGU AGUGmGmGmUmAmAmAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCmAmUmCmAAAGNNNNNNNNNNNNNNNNNmN*mN*mN 2950 174 (v4) mA*mC*mU*mGmGmCmGmCUUUUmAmUmCmUmGmAmUUACUUUGmAmGmAmGmCmCmAmUmCmAmCmCAGCGAmCmUAUmGmUmCmGUA GUGmGmGmUmAmAmAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCmAmUCAAAGNNNNNNNNNNNNNNNNNmN*mN*mN 2951 174 (v5) mA*mC*mU*GGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGAmCmUAUmGmUmCmGUAGUGGGUAAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCAUCAAAGNNNNNNNNNNNNNNNNNmN*mN*mN 2952 174 (v6) mA*mC*mU*GGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCAUCAAAGNNNNNNNNNNNNNNNNNmN*mN*mN 2953 174 (v7) mA*mC*mU*GGmCGmCmUUUUAmUmCUGAUUACUUUGmAmGAGCCmAmUmCmAmCCAGCmGmAmCmUAUmGmUmCmGUAGUGG mGmUAmAmAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCmAmUCAAAGNNNNNNNNNNNNNNN*mN*mN*mN 2954 174 (v8) mA*mC*mU*GGCGCUUUUAUCUGAUUACUUUGAGAGCCmAmUmCmAmCCAGCmGmAmCmUAUmGmUmCmGUAGUGGmGmU mAmAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCmAmUCAAAGNNNNNNNNNNNNNNN*mN*mN*mN 2955 174 (v9) mA*mC*mU*GGmCmGCmUUUUAmUmCUGAUUACUUUGmAmGAGCCAUCACCAGCmGmAmCmUAUmGmUmCmGUAGUGGGGUAAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCAUCAAAGNNNNNNNNNNNNNNN*mN*mN*mN 2956 235 (v0) ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCCGUAAGAGGCAUCAGAGNNNNNNNNNNNNNNNNNN 2957 235 (v1) mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAGNNNNNNNNNNNNNNNNNmN*mN*mN 2958 235 (v2) mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAGNNNNNNNNNNNNNNNNNN*mU*mU*mU 2959 235 (v3) mA*mC*mU*mGmGmCmGmCmUmUmCmUmAmUmCmUmGmAmUUACUCUGmAmGmCmGmCmCmAmUmCmAmCmCAGCGAmCmUAUmGmUmCmGUAGUGmGmGmU mAmAmAmGmCmCmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCmAmUmCmAGAGNNNNNNNNNNNNNNNNNmN*mN*mN 2960 235 (v4) mA*mC*mU*mGmGmCmGmCUUCUmAmUmCmUmGmAmUUACUCUGmAmGmCmGmCmCmAmUmCmAmCmCAGCGAmCmUAUmGmUmCmGUAGUGmGmGmUm AmAmAmGmCmCmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCmAmUCAGAGNNNNNNNNNNNNNNNNNmN*mN*mN 2961 235 (v5) mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGAmCmUAUmGmUmCmGUAGUGGGGUmAmAmAmGmCmCm GmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCAUCAGAGNNNNNNNNNNNNNNNmN*mN*mN 2962 235 (v6) mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUmAmAmAmGmCmCmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCAUCAGAGNNNNNNNNNNNNNNNmN*mN*mN 2963 235 (v7) mA*mC*mU*GGmCGmCmUUCUAmUmCUGAUUACUCUGmAmGCGCCmAmUmCmAmCCAGCmGmAmCmUAUmGmUmCmGUAGUGGGmGmUmAmAAm GmCmCmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCmAmUCAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2964 235 (v8) mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCmAmUmCmAmCCAGCmGmAmCmUAUmGmUmCmGUAGUGGmGmUmAmAAmGmCm CmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCmAmUCAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2965 235 (v9) mA*mC*mU*GGmCGmCmUUCUAmUmCUGAUUACUCUGmAmGCGCCAUCACCAGCmGmAmCmUAUmGmUmCmGUAGUGGGUAAAmGmCm CmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCAUCAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2966 316 (v0) ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGNNNNNNNNNNNNNNNNNN 2967 316 (v1) mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2968 316 (v2) mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGNNNNNNNNNNNNNNNNNN*mU*mU*mU 2969 316 (v3) mA*mC*mU*mGmGmCmGmCmUmUmCmUmAmUmCmUmGmAmUUACUCUGmAmGmCmGmCmCmAmUmCmAmCmCAGCGAmCmUAUmGmUmCmGU AGUGmGmGmUmAmAmAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCmAmUmCmAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2970 316 (v4) mA*mC*mU*mGmGmCmGmCUUCUmAmUmCmUmGmAmUUACUCUGmAmGmCmGmCmCmAmUmCmAmCmCAGCGAmCmUAUmGmUmCmGUA GUGmGmGmUmAmAmAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCmAmUCAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2971 316 (v5) mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGAmCmUAUmGmUmCmGUAGUGGGGUAAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCAUCAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2972 316 (v6) mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCAUCAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2973 316 (v7) mA*mC*mU*GGmCGmCmUUCUAmUmCUGAUUACUCUGmAmGCGCCmAmUmCmAmCCAGCmGmAmCmUAUmGmUmCmGUAGUGG mGmUmAmAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCmAmUCAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2974 316 (v8) mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCmAmUmCmAmCCAGCmGmAmCmUAUmGmUmCmGUAGUGGmGmU mAmAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCmAmUCAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2975 316 (v9) mA*mC*mU*GGmCGmCmUUCUAmUmCUGAUUACUCUGmAmGCGCCAUCACCAGCmGmAmCmUAUmGmUmCmGUAGUGGGGUAAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCAUCAGAGNNNNNNNNNNNNNNN*mN*mN*mN 2976 Table 23 : Sequences of gRNAs and their overview of the different chemical modifications analyzed in this example ( indicated by model number ) . Chemical modifications : * = phosphorothioate bond ; m = 2'OMe modification gRNA ID (Scaffold variant-spacer) Target gRNA sequences SEQ ID NO: 174-6.7 (v0) Human PCSK9 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGUCCUGGCUUCCUGGUGAAGA 3145 174-6.7 (v1) Human PCSK9 mA*mC*mU*GGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGUCCUGGCUUCCUGGUGAmA*mG*mA 3074 174-6.8 (v0) Human PCSK9 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGUGGCUUCCUGGUGAAGAUGA 3146 174-6.8 (v1) Human PCSK9 mA*mC*mU*GGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGUGGCUUCCUGGUGAAGAmU*mG*mA 3147 174-7.9 (v0) Human B2M ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGGUGUAGUACAAGAGAUAGAA 3148 174-7.9 (v1) Human B2M mA*mC*mU*GGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGGUGUAGUACAAGAGAUAmG*mA*mA 3149 316-6.7 (v0) Human PCSK9 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGUCCUGGCUUCCUGGUGAAGA 3150 316-6.7 (v1') Human PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGUCCUGGCUUCCUGGUGAmA*mG*mA 3151 316-6.8 (v0) Human PCSK9 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGUGGCUUCCUGGUGAAGAUGA 3152 316-6.8 (v1') Human PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGUGGCUUCCUGGUGAAGAmU*mG*mA 3153 316-7.9 (v0) Human B2M ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGGUGUAGUACAAGAGAUAGAA 3154 316-7.9 (v1') Human B2M mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGGUGUAGUACAAGAGAUAmG*mA*mA 3155 174-7.37 (v0) Human B2M ACUGGCGCUUUUAUCUgAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAgUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGGGCCGAGAUGUCUCGCUC 3156 174-7.37 (v1*) Human B2M mA*mC*mU*GGCGCUUUUAUCUgAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAgUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGGGCCGAGAUGUCUCG*mC*mU*mC 3157 235-6.7 (v0) Human PCSK9 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCCGUAAGAGGCAUCAGAGUCCUGGCUUCCUGGUGAAGA 3158 235-6.7 (v1) Human PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAGUCCUGGCUUCCUGGUGAmA*mG*mA 3159 235-6.7 (v2) Human PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAGUCCUGGCUUCCUGGUGAAGAU*mU*mU*mU 3160 235-6.7 (v3) Human PCSK9 mA*mC*mU*mGmGmCmGmCmUmUmCmUmAmUmCmUmGmAmUUACUCUGmAmGmCmGmCmCmAmUmCmAmCmCAGCGAmCmUAUmGmUmCmGUAGUGmGmGmU mAmAmAmGmCmCmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCmAmUmCmAGAGUCCUGGCUUCCUGGUGAmA*mG*mA 3161 235-6.7 (v4) Human PCSK9 mA*mC*mU*mGmGmCmGmCUUCUmAmUmCmUmGmAmUUACUCUGmAmGmCmGmCmCmAmUmCmAmCmCAGCGAmCmUAUmGmUmCmGUAGUGmGmGmUm AmAmAmGmCmCmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCmAmUCAGAGUCCUGGCUUCCUGGUGAmA*mG*mA 3162 235-6.7 (v5) Human PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGAmCmUAUmGmUmCmGUAGUGGGGUmAmAmAmGmCmCm GmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCAUCAGAGUCCUGGCUUCCUGGUGAmA*mG*mA 3163 235-6.7 (v6) Human PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUmAmAmAmGmCmCmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCAUCAGAGUCCUGGCUUCCUGGUGAmA*mG*mA 3164 235-6.8 (v0) Human PCSK9 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAGUGGCUUCCUGGUGAAGAUGA 3165 235-6.8 (v1) Human PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAGUGGCUUCCUGGUGAAGAmU*mG*mA 3166 235-6.8 (v2) Human PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAGUGGCUUCCUGGUGAAGAUGA*mU*mU*mU 3167 235-6.8 (v3) Human PCSK9 mA*mC*mU*mGmGmCmGmCmUmUmCmUmAmUmCmUmGmAmUUACUCUGmAmGmCmGmCmCmAmUmCmAmCmCAGCGAmCmUAUmGmUmCmGUAGUGmGmGmU mAmAmAmGmCmCmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCmAmUmCmAGAGUGGCUUCCUGGUGAAGAmU*mG*mA 3168 235-6.8 (v4) Human PCSK9 mA*mC*mU*mGmGmCmGmCUUCUmAmUmCmUmGmAmUUACUCUGmAmGmCmGmCmCmAmUmCmAmCmCAGCGAmCmUAUmGmUmCmGUAGUGmGmGmUm AmAmAmGmCmCmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCmAmUCAGAGUGGCUUCCUGGUGAAGAmU*mG*mA 3169 235-6.8 (v5) Human PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGAmCmUAUmGmUmCmGUAGUGGGGUmAmAmAmGmCmCm GmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCAUCAGAGUGGCUUCCUGGUGAAGAmU*mG*mA 3170 235-6.8 (v6) Human PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUmAmAmAmGmCmCmGmCmUmUmAmCmGmGmAmCmUmUmCmGmGmUmCmCmGmUmAmAmGmAmGmGmCAUCAGAGUGGCUUCCUGGUGAAGAmU*mG*mA 3171 316-27.107 (v0) Mouse PCSK9 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGCUGGCUUCUUGGUGAAGAUG 3172 316-27.107 (v1) Mouse PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGCUGGCUUCUUGGUGAAG*mA*mU*mG 3173 316-27.107 (v7) Mouse PCSK9 mA*mC*mU*GGmCGmCmUUCUAmUmCUGAUUACUCUGmAmGCGCCmAmUmCmAmCCAGCmGmAmCmUAUmGmUmCmGUAGUGG mGmUmAmAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCmAmUCAGAGCUGGCUUCUUGGUGAAG*mA*mU*mG 3174 316-27.107 (v8) Mouse PCSK9 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCmAmUmCmAmCCAGCmGmAmCmUAUmGmUmCmGUAGUGGmGmU mAmAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCmAmUCAGAGCUGGCUUCUUGGUGAAG*mA*mU*mG 3175 316-27.107 (v9*) Mouse PCSK9 mA*mC*mU*GGmCGmCmUUCUAmUmCUGAUUACUCUGmAmGCGCCAUCACCAGCmGmAmCmUAUmGmUmCmGUAGUGGG UAAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCAUCAGAGCUGGCUUCUUGGUGAA*mG*mA*mU*mG 3176 174-35.2 (v0) ROSA26 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGAGAAGAUGGGCGGGAGUCUU 3177 174-35.2 (v2) ROSA26 mA*mC*mU*GGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAGAGAAGAUGGGCGGGAGUCUU*mU*mU*mU 3178 316-35.2 (v0) ROSA26 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGAGAAGAUGGGCGGGAGUCUU 3179 316-35.2 (v1) ROSA26 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAGAGAAGAUGGGCGGGAGU*mC*mU*mU 3180 316-35.2 (v5) ROSA26 mA*mC*mU*GGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGAmCmUAUmGmUmCmGUAGUGGGGUAAAmGmCmUmCmCmCmUmCmUmUmCmGmGmAmGmGmGmAmGmCAUCAGAGAGAAGAUGGGCGGGAGU*mC*mU*mU 3181 Note that gRNAs labeled with v1' design have one less phosphorothioate bond at the 3' end of the gRNA. gRNAs labeled with v1* have one more phosphorothioate bond at the 3' end of the gRNA. gRNAs labeled with v9* have one more phosphorothioate bond at the 3' end of the gRNA. Biochemical Signs of gRNA Activity:

Target DNA oligonucleotides with a fluorescent moiety on the 5' end were commercially purchased (sequences listed in Table 24). Double-stranded DNA (dsDNA) targets were formed by mixing the oligonucleotides at a 1:1 ratio in 1× lysis buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCl ₂ ), followed by heating to 95°C for 10 minutes, and then cooling the solution to room temperature. CasX ribonucleoprotein (RNP) was reconstituted with CasX 491 and the designated gRNA in 1× lysis buffer at a final concentration of 1 µM and a 1.2-fold excess of the designated gRNA. RNPs were allowed to form at 37°C for 10 minutes.

The effects of various structural and chemical modifications of the gRNA scaffold on the cleavage rate of CasX 491 RNP were determined. Cleavage reactions were prepared with a final RNP concentration of 200 nM and a final target concentration of 10 nM, and the reactions were performed at 16°C and initiated by the addition of labeled target DNA substrate (Table 24). Aliquots of the reactions were taken at 0.25, 0.5, 1, 2, 5, and 10 minutes and quenched by adding equal volumes of 95% formamide and 20 mM EDTA. Samples were denatured at 95°C for 10 minutes and resolved on 10% urea-PAGE gels. Gels were imaged on a Typhoon ^™ laser-scanner platform and quantified using ImageQuant ^™ TL 8.2 image analysis software (Cytiva ^™ ). The apparent first-order rate constant for off-target strand cleavage (k _cleavage ) was determined for each CasX:gRNA combination. _­ ).

To determine the fraction of competence formed by each gRNA, cleavage reactions were prepared with a final RNP concentration of 100 nM and a final target concentration of 100 nM. Reactions were performed at 37°C and initiated by adding labeled target substrate (Table 24). Aliquots were taken at 0.5, 1, 2, 5, 10, and 30 minutes and quenched by adding equal volumes of 95% formamide and 25 mM EDTA. Samples were denatured by heating at 95°C for 10 minutes and resolved on 10% urea-PAGE gels. Gels were imaged and quantified as above. It is assumed that CasX acts as a single turnover enzyme under the assay conditions, as indicated by the observation that substoichiometric amounts of enzyme will fail to cleave target substrates in excess of stoichiometric amounts even on extended time scales, but will instead approach a plateau proportional to the amount of enzyme present. Therefore, the fraction of target substrate cleaved by equimolar amounts of RNPs on long time scales will indicate the fraction of RNPs that are properly formed and active for cleavage. The cleavage traces were fit with a two-phase rate model, as the cleavage reaction deviates significantly from a single phase under this concentration regime. Each fitted plateau was determined and reported in Table 27 as the fractional activity of each RNP. Table 24 : Sequences of target DNA substrate oligonucleotides with a fluorescent moiety on the 5' end for biochemical characterization of gRNA activity. /700/ = IRDye700; /800/ = IRDye800 DNA Substrate sequence 6.7/6.8 Target Stock (SEQ ID NO: 3182) /700/catgtcttccatggccttcttcctggcttcctggtgaagatgagtggcgacctgctggag 6.7/6.8 target stock placement (SEQ ID NO: 3183) /800/ctccagcaggtcgccactcatcttcaccaggaagccaggaagaaggccatggaagacatg In vitro transcription of CasX mRNA:

DNA templates encoding CasX 491 for in vitro transcription (see Table 25 for coding sequences) were generated by PCR using a forward primer containing a T7 promoter followed by agarose gel extraction of the appropriate size. A final concentration of 25 ng/µL of template DNA was used in each in vitro transcription reaction, which was performed according to the manufacturer's recommended protocol with slight modifications. After incubation of the in vitro transcription reactions for 2 to 3 hours at 37°C with CleanCap® AG and N1-methyl-pseudouridine, the template DNA was DNase digested and column-based purified using the Zymo RNA miniprep kit. The poly(A) tail was added using E. coli poly(A) polymerase according to the manufacturer's protocol, followed by column-based purification as described above. The poly(A)-tailed ex vivo transcribed RNA was dissolved in RNase-free water, analyzed for integrity on an Agilent TapeStation, and flash frozen and subsequently stored at -80°C. Table 25 : Coding sequences of CasX mRNA molecules evaluated in this example * CasX 491 mRNA ID Component (ID) DNA Sequence SEQ ID NO: CasX 491 mRNA #1 5'UTR GACCGGCCGCCACC 3082 START codon + c-MYC NLS + linker ATGGCCCCAGCGGCCAAACGGGTGAAGCTGGACTCTAGA 3184 CasX 491 CAAGAGATCAAGAGAATCAACAAGATCAGAAGGAGACTGGTCAAGGACAGCAACACAAAGAAGGCCGGCAAGACAGGCCCCATGAAAACCCTGCTCGTCAGAGTGATGACCCCTGACCTGAGAGAGCGGCTGGAAAACCTGAGAAAGAAGCCCGAGAACATCCCTCAGCCTATCAGCAACACC AGCAGGGCCAACCTGAACAAGCTGCTGACCGACTACACCGAGATGAAGAAAGCCATCCTGCACGTGTACTGGGAAGAGTTCCAGAAAGACCCCGTGGGCCTGATGAGCAGAGTTGCTCAGCCTGCCAGCAAGAAGATCGACCAGAACAAGCTGAAGCCCGAGATGGACGAGAAGGGCAATCTG ACCACAGCCGGCTTTGCCTGCTCTCAGTGTGGCCAGCCTCTGTTCGTGTACAAGCTGGAACAGGTGTCCGAGAAAGGCAAGGCCTACACCAACTTCGGCAGATGTAACGTGGCCGAGCACGAGAAGCTGATTCTGCTGGCCCAGCTGAAACCTGAGAAGGACTCTGATGAGGCCGTGACC TACAGCCTGGGCAAGTTTGGACAGAGAGCCCTGGACTTCTACAGCATCCACGTGACCAAAGAAAGCACACACCCCGTGAAGCCCCTGGCTCAGATCGCCGGCAATAGATACGCCTCTGGACCTGTGGGCAAAGCCCTGTCCGATGCCTGCATGGGAACAATCGCCAGCTTCCTGAGCAAGTAC CAGGACATCATCGAGCACCAGAAGGTGGTCAAGGGCAACCAGAAGAGACTGGAAAGCCTGAGGGAGCTGGCCGGCAAAGAGAACCTGGAATACCCCAGCGTGACCCTGCCTCCTCAGCCTCACACAAAAGAAGGCGTGGACGCCTACAACGAAGTGATCGCCAGAGTGAGAATGTGGGTC AACCTGAACCTGTGGCAGAAGCTGAAACTGTCCAGGGACGACGCCAAGCCTCTGCTGAGACTGAAGGGCTTCCCTAGCTTCCCTCTGGTGGAAAGACAGGCCAATGAAGTGGATTGGTGGGACATGGTCTGCAACGTGAAGAAGCTGATCAACGAGAAGAAAGAGGATGGCAAGGTTTTCTGG CAGAACCTGGCCGGCTACAAGAGACAAGAAGCCCTGAGGCCTTACCTGAGCAGCGAAGAGGACCGGAAGAAGGGCAAGAAGTTCGCCAGATACCAGCTGGGCGACCTGCTGCTGCACCTGGAAAAGAAGCACGGCGAGGACTGGGGCAAAGTGTACGATGAGGCCTGGGAGAGAATCGACAAG AAGGTGGAAGGCCTGAGCAAGCACATTAAGCTGGAAGAGGAAAGAAGGAGCGAGGACGCCCAATCTAAAGCCGCTCTGACCGATTGGCTGAGAGCCAAGGCCAGCTTTGTGATCGAGGGCCTGAAAGAGGCCGACAAGGACGAGTTCTGCAGATGCGAGCTGAAGCTGCAGAAGTGGTACGGCG ATCTGAGAGGCAAGCCCTTCGCCATTGAGGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCAGTACAACTGCGCCTTCATTTGGCAGAAAGACGGCGTCAAGAAACTGAACCTGTACCTGATCATCAATTACTTCAAAGGCGGCAAGCTGCGGTTCAAGAAGATCAAACCCGAGG CCTTCGAGGCTAACAGATTCTACACCGTGATCAACAAAAAGTCCGGCGAGATCGTGCCCATGGAAGTGAACTTCAACTTCGACGACCCCAACCTGATTATCCTGCCTCTGGCCTTCGGCAAGAGACAGGGCAGAGAGTTCATCTGGAACGATCTGCTGAGCCTGGAAACCGGCTCTCTGAAGC TGGCCAATGGCAGAGTGATCGAGAAAACCCTGTACAACAGGAGAACCAGACAGGACGAGCCTGCTCTGTTTGTGGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGCAACATCAAGCCCATGAACCTGATCGGCGTGGACCGGGGCGAGAATATCCCTGCTGTGATCGCCCTGACAG ACCCTGAAGGATGCCCACTGAGCAGATTCAAGGACTCCCTGGGCAACCCTACACACATCCTGAGAATCGGCGAGAGCTACAAAGAGAAGCAGAGGACAATCCAGGCCAAGAAAGAGGTGGAACAGCGCAGAGCCGGCGGATACTCTAGGAAGTACGCCAGCAAGGCCAAGAATCTGGCCGACGA CATGGTCCGAAACACCGCCAGAGATCTGCTGTACTACGCCGTGACACAGGACGCCATGCTGATCTTCGAGAATCTGAGCAGAGGCTTCGGCCGGCAGGGCAAGAGAACCTTTATGGCCGAGAGGCAGTACACCAGAATGGAAGATTGGCTCACAGCTAAACTGGCCTACGAGGGACTGAGCAA GACCTACCTGTCCAAAACACTGGCCCAGTATACCTCCAAGACCTGCAGCAATTGCGGCTTCACCATCACCAGCGCCGACTACGACAGAGTGCTGGAAAAGCTCAAGAAAACCGCCACCGGCTGGATGACCACCATCAACGGCAAAGAGCTGAAGGTTGAGGGCCAGATCACCTACTACAACAG GTACAAGAGGCAGAACGTCGTGAAGGATCTGAGCGTGGAACTGGACAGACTGAGCGAAGAGAGCGTGAACAACGACATCAGCAGCTGGACAAAGGGCAGATCAGGCGAGGCTCTGAGCCTGCTGAAGAAGAGGTTTAGCCACAGACCTGTGCAAGAGAAGTTCGTGTGCCTGAACTGCGGCTT CGAGACACACGCCGATGAACAGGCTGCCCTGAACATTGCCAGAAGCTGGCTGTTCCTGAGAAGCCAAGAGTACAAGAAGTACCAGACCAACAAGACCACCGGCAACACCGACAAGAGGGCCTTTGTGGAAAACCTGGCAGAGCTTCTACAGAAAAAAGCTGAAAGAAGTCTGGAAGCCCGCCGTG 3185 Linker + c-MYC NLS GGCTCCCCCGCCGCGAAGCGAGTGAAACTGGACGGATCCGGC 3186 P2A mScarlet + STOP code GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGATGGTATCAAAGGGTGAGGCGGTCATTAAGGAATTCATGCGGTTCAAAGTTCACATGGAGGGGAGTATGAATGGTCACGAATTTGAGATCGAGGGTGAGGGAGAGGGTAGACCTTACGAAGGCACCCAGACA GCAAAACTCAAAGTCACTAAGGGCGGCCCACTCCCTTTCTCATGGGATATACTGAGTCCTCAATTTATGTATGGCTCCCGGGCCTTTATCAAGCATCCAGCCGACATACCCGATTACTATAAACAGTCATTCCCCGAGGGATTCAAATGGGAGAGAGTGATGAATTTTGAAGACGGGGGTGCCGTGACC GTAACTCAAGATACCTCTTTGGAAGATGGTACCCTCATTTACAAGGTTAAGCTTAGAGGAACTAATTTCCCACCAGACGGACCCGTTATGCAGAAAAAAACCATGGGTTGGGAGGCTAGCACCGAACGGCTGTATCCAGAGGATGGAGTTTTGAAAGGTGATATTAAGATGGCCCTGCGCCTGAAAGAC GGAGGCGGTACCTCGCAGATTTCAAAACTACCTACAAAGCTAAGAAACCGGTCCAAATGCCGGGTGCTTATAACGTCGATAGGAAGCTGGATATAACATCACACAACGAAGACTATACGGTTGTCGAACAGTATGAGCGATCTGAGGGGAGGCACAGCACGGGCGGGATGGATGAGCTTTACAAGTGA 3187 CasX 676 mRNA #2 5'UTR AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC 3047 START codon + c-MYC NLS ATGGCCCCTGCTGCCAAGAGAGTGAAGCTGGATAGCAGA 3133 CasX 676 See the sequence in Table 19 3134 c-MYC NLS + STOP codon GGAAGCCCCGCTGCCAAGAGAGTGAAGCTGGACTAATAGATAA 3135 3'UTR GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG 3055 XbaI restriction site (partial) TCTAG 3056 Poly(A) tail AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3057 * Components are listed in 5' to 3' sequence within the construct. Delivery of gRNA and CasX mRNA in vitro via transfection:

The editing of the PCSK9 locus and the subsequent effect on secreted PCSK9 levels were assessed for conditions using CasX 491 mRNA co-delivered with PCSK9 targeting gRNA with scaffold variant 174 compared to conditions using PCSK9 targeting gRNA with scaffold variant 316. 100 ng of in vitro transcribed mRNA encoding CasX 491 with P2A and mScarlet fluorescein was transfected into HepG2 cells with gRNA 174-6.7, 174-6.8, 316-6.7 and version 1 (v1) of 316-6.8 using lipofectamine (see Table 23). After changing the medium, the following were collected 28 hours after transfection: 1) transfected cells were collected to assess editing at the PCSK9 locus by next generation sequencing (NGS); and 2) culture supernatants were collected to measure secreted PCSK9 protein levels by ELISA. For editing analysis by NGS, amplicon was amplified from 200 ng of extracted gDNA with a set of primers targeting the PCSK9 locus and processed for sequencing (described below). Secreted PCSK9 levels in culture supernatants were also analyzed using a fluorescence resonance energy transfer-based immunoassay from CISBio according to the manufacturer's instructions. Here, scaffold 174 and a gRNA targeting spacer 7.37 (v0; see Table 23) of the endogenous B2M (β-2-microglobulin) locus were used as a non-targeting (NT) control. These results are shown in FIG. 15 .

To compare the editing efficacy of unmodified version 0 (v0) and chemically modified version 1 (v1) of B2M targeting gRNA, approximately 6E4 HepG2 hepatocytes were seeded per well of a 96-well plate. 24 hours later, the seeded cells were co-transfected with lipofectamine and 100 ng of ex vivo transcribed mRNA encoding CasX 491 and different doses (1, 5 or 50 ng) of v0 or v1 of B2M targeting gRNA containing scaffold variant 174 and spacer 7.37 (see Table 23). Six days after transfection, cells were harvested for immunostaining of B2M-dependent HLA proteins, followed by flow cytometry analysis of B2M protein expression using an Attune ^™ NxT flow cytometer. These results are shown in Figure 16.

The effects of v1 to v6 variants of chemically modified PCSK9 targeting gRNAs (Table 23) on editing efficacy and subsequent effects on secreted PCSK9 levels were assessed. Briefly, 100 ng of in vitro transcribed mRNA encoding CasX variant 491 and P2A and mScarlet fluorescent protein were transfected into HepG2 cells with 50 ng of the specified chemically modified gRNA using lipofectamine. After changing the medium, the following were collected 28 hours after transfection: 1) transfected cells were collected as described above to assess editing at the PCSK9 locus by NGS; and 2) culture supernatants were collected as described above to measure secreted PCSK9 protein levels by ELISA. Here, B2M targeting gRNA was used as a non-targeting control. These results are shown in Table 28. NGS Processing and Analysis:

Genomic DNA (gDNA) from the collected cells was extracted using the Zymo Quick-DNA Miniprep Plus kit according to the manufacturer's instructions. Targeted amplicon was formed by amplifying the region of interest of approximately 50 to 200 ng of extracted gDNA with a set of primers targeting the human PCSK9 locus. These gene-specific primers contain additional sequences at the 5′ end to introduce Illumina read 1 and 2 sequences. In addition, they contain a 16-nucleotide random sequence that serves as a unique molecular identifier (UMI). The quality and quantification of the amplicon was assessed using the Fragment Analyzer DNA analyzer kit (Agilent, dsDNA 35-1500 bp). Amplicon was sequenced on an Illumina MiSeq™ according to the manufacturer's instructions. The raw fastq sequencing files were processed by trimming the quality and adapter sequences and merging reads 1 and 2 into a single insert sequence; the insert sequence was then analyzed by the CRISPResso2 (v 2.0.29) program. The percentage of modified reads in the window around the 3' end of the spacer was determined. For each, the activity of the CasX molecule was quantified as the total percentage of reads containing insertions, substitutions and/or deletions anywhere within this window. Formulation of Lipid Nanoparticles (LNPs):

CasX mRNA and gRNA were encapsulated into LNPs using GenVoy-ILM ^™ lipids on a Precision NanoSystems Inc. (PNI) Ignite ^™ Benchtop system according to the manufacturer's instructions. GenVoy-ILM ^™ lipids were manufactured by PNI and had a composition of ionizable lipid: DSPC: cholesterol: stabilizer at 50:10:37.5:2.5 mol%.

Briefly, to prepare LNPs, CasX mRNA and gRNA were diluted in PNI preparation buffer (pH 4.0) at equal mass ratios. GenVoy-ILM ^™ was diluted 1:1 in absolute ethanol. mRNA/gRNA co-formulation was performed using a predetermined N/P ratio. RNA and lipids were passed through a PNI laminar flow filter cartridge on a PNI Ignite ^™ Benchtop system at a predetermined flow rate ratio (RNA:Genvoy-ILM ^™ ). After preparation, LNPs were diluted in PBS (pH 7.4) to reduce the ethanol concentration and increase the pH, thereby improving the stability of the particles. Buffer exchange of mRNA/sgRNA-LNPs was achieved by dialysis into PBS (pH 7.4) at 4°C using a 10k Slide-A-Lyzer™ Dialysis Cassette (Thermo Scientific™). After dialysis, mRNA/gRNA-LNPs were concentrated to > 0.5 mg/mL using a 100 kDa Amicon®-Ultra centrifugal filter (Millipore) and subsequently filter-sterilized. The formulated LNPs were analyzed on a Stunner (Unchained Labs) to determine their diameter and polydispersity index (PDI). Encapsulation efficiency and RNA concentration were determined by RiboGreen ^™ analysis using Invitrogen's Quant-iT ^™ RiboGreen ^™ RNA Assay Kit. In each of the experiments described herein, LNPs were used to deliver CasX mRNA and gRNA to target cells and tissues. In vitro delivery of LNPs encapsulating CasX mRNA and targeting gRNA:

Approximately 50,000 HepG2 cells were seeded per well in a 96-well plate in DMEM/F-12 medium containing 10% FBS and 1% PenStrep. The next day, the seeded cells were treated with varying concentrations of LNPs, starting at 250 ng, prepared in six 2-fold serial dilutions. These LNPs were formulated to encapsulate CasX 491 mRNA and gRNA targeting B2M (v1; see Table 23) incorporating scaffold variants 174 or 316 and spacer 7.9. The medium was changed 24 hours after LNP treatment, and the cells were cultured for an additional six days before being harvested for extraction of gDNA for assessment of editing at the B2M locus by NGS, and HLA immunostaining followed by flow cytometry analysis of B2M protein expression using an Attune NxT flow cytometer. Briefly, for editing assessment, amplicon was amplified from 200 ng of gDNA extracted with primers targeting the human B2M locus and processed by NGS using the method described below. The results of these analyses are shown in Figures 17 and 18.

Approximately 20,000 mouse Hepa1-6 cells were seeded per well in a 96-well plate. The next day, the seeded cells were treated with varying concentrations of LNPs, starting at 1000 ng, prepared in eight 2-fold serial dilutions. These LNPs were formulated to encapsulate CasX 676 mRNA #2 (see Table 25) and a gRNA targeting ROSA26 (v1 or v5; see Table 23) incorporating scaffold variant 316 and spacer 35.2. The medium was changed 24 hours after treatment with LNPs, and the cells were cultured for an additional seven days before being harvested for extraction of gDNA for evaluation of editing at the ROSA26 locus by NGS. Briefly, amplicon was amplified from extracted gDNA with primers targeting the mouse ROSA26 locus and processed by NGS using a similar method as described below. The results of this experiment are shown in Figure 19. In vivo delivery of LNPs encapsulating CasX mRNA and targeting gRNA:

To assess the effects of using v1 and v5 scaffolds 316 in vivo, CasX 676 mRNA #2 (see Table 25) and gRNA targeting ROSA26 (v1 or v5; see Table 23) using scaffold 316 and spacer 35.2 were encapsulated in the same LNP using a 1:1 mRNA:gRNA mass ratio. The formulated LNPs were exchanged with PBS for intravitreal injection. Briefly, LNPs were administered intravenously via the posterior orbital sinus to 4-week-old C57BL/6 mice. Mice were observed for five minutes after injection to ensure recovery from anesthesia and then placed in cages. Initial, non-injected animals were used as experimental controls. Six days after administration, mice were sacrificed and liver tissue was collected for gDNA extraction using the Zymo Research Quick DNA/RNA Miniprep kit according to the manufacturer's instructions. The target amplicon was then amplified from the extracted gDNA using a set of primers targeting the mouse ROSA26 locus and processed using a similar method as described below to evaluate the edit by NGS. The results of this experiment are shown in Figure 20.

To compare the effects of scaffold 316 using v7, v8, and v9 on editing of the PCSK9 locus in vivo, CasX 676 mRNA #1 (see Table 26 for sequence) and PCSK9 targeting gRNAs (v1, v7, v8, or v9; see Table 23) using scaffold 316 and spacer 27.107 were encapsulated in the same LNP using a 1:1 mRNA:gRNA mass ratio for each gRNA. LNPs were administered retro-orbitally to 6-week-old C57BL/6 mice as described above, and mice were sacrificed seven days after injection to collect liver tissue for extraction of gDNA for assessment of editing at the PCSK9 locus by NGS. The results of this experiment are shown in Figure 21. Table 26 : Coding sequence of CasX 676 mRNA #1 molecule CasX ID Component (ID) describe DNA Sequence SEQ ID NO: CasX 676 mRNA #1 5'UTR HbA ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC 3188 START codon + c-MYC NLS ATGGCCCCTGCTGCCAAGAGAGTGAAGCTGGATAGCAGA 3133 CasX 676 See the sequence in Table 19 3134 c-MYC NLS + STOP codon GGAAGCCCCGCTGCCAAGAGAGTGAAGCTGGACTAATAGATAA 3135 3'UTR HbA GCTGGAGCCTCGGTGGCCATGCTTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA 3189 Poly(A) tail AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3057 *Each component is listed in the order of 5' to 3' in the construct. Results: Evaluation of the effects of various chemical modifications on gRNA activity:

Several studies involving Cas9 have demonstrated that chemical modification of gRNAs can significantly improve editing activity when delivered with Cas9 mRNA. After delivery of Cas9 mRNA and gRNA into target cells, unprotected gRNAs are susceptible to degradation during the mRNA translation process. Adding chemical modifications such as 2'O-methyl (2'OMe) groups and phosphorothioate bonds can reduce the susceptibility of gRNAs to cellular RNases, but may also disrupt gRNA folding and its interaction with CRISPR-Cas proteins. Given the lack of structural similarity between CasX and Cas9 and their respective gRNAs, appropriate chemical modification profiles must be designed and validated de novo. Using the published structure of wild-type CasX from Deltaproteobacteria (PDB codes 6NY1, 6NY2, and 6NY3) as a reference, residues that seemed likely to be suitable for modification were selected. However, the published structures are those of wild-type CasX orthologs and gRNAs, which are different from the species used as the basis for the engineered variants presented herein, and they also lack the resolution to confidently determine the interactions between the protein side chains and the RNA backbone. These limitations bring great uncertainty to determining which nucleotides can be safely modified. Therefore, six chemical modification profiles (represented as patterns) were designed for initial testing, and these six profiles are shown in Figures 10 and 11. The v1 profile was designed as a simple end-protection structure in which the first and last three nucleotides were modified with 2'OMe and phosphorothioate bonds. In the v2 profile, a 3'UUU tail was added to mimic the termination sequence used in cellular transcription systems, and the modified nucleotides were moved outside the spacer region involved in target recognition. The v3 profile included end-protection as in v1, and the addition of 2'OMe modifications on all nucleotides identified as potentially modifiable based on structural analysis. The v4 profile was modeled based on v3, but with all modifications in the triple helical region removed, as this structure is predicted to be more sensitive to any perturbations of the RNA helical structure and backbone flexibility. The v5 profile maintains chemical modifications in both the scaffold stem and the extension stem regions, whereas the v6 profile has modifications only in the extension stem. The extension stem is the region of the RNP that is fully exposed to solvents and which can be replaced by other hairpin structures and therefore may be relatively insensitive to chemical modifications.

Minimally modified v1 gRNAs were first evaluated relative to unmodified gRNAs (v0) to determine the potential benefit of such chemical modifications on editing when gRNAs were co-delivered with CasX mRNA to target cells. Modified (v1) and unmodified (v0) B2M -targeting gRNAs with spacer 7.37 were co-transfected with CasX mRNA into HepG2 cells, and editing at the B2M locus was measured by flow cytometry to detect loss of B2M-dependent HLA complex surface presentation ( FIG. 16 ). The data showed that the use of v1 gRNA resulted in a much greater loss of B2M expression compared to the levels observed at multiple doses of v0 gRNA, confirming that terminal modifications of the gRNA increase CasX-mediated editing activity following delivery of CasX mRNA and gRNA.

A broader set of gRNA chemical modification profiles was assessed using PCSK9 targeting gRNAs utilizing scaffold variant 235 and spacers 6.7 and 6.8 to determine whether additional chemical modifications would be able to support the formation of active RNPs. The in vitro cleavage assays described above were performed to determine the k _cleavage and fractional capacity of these engineered gRNAs with various chemical modification profiles. The results from these in vitro cleavage assays are shown in Table 27. The data indicate that gRNAs with v3 profiles do not exhibit activity, indicating that adding some chemical modifications significantly interferes with RNP formation or activity. Adding v4 chemical modifications resulted in reasonable cleavage rates in excess RNP conditions, but exhibited extremely low fractional capacity. The difference between v3 and v4 modifications demonstrates that modification of the triple helical region prevents any active RNP formation, either due to a failure of the gRNA to fold properly or disruption of the gRNA-protein interaction. The reduction in fractional competence caused by the addition of the v4 modification suggests that, although the gRNA is able to successfully assemble with the CasX protein to form cleavage-competent RNPs, the vast majority of gRNAs fold incorrectly, or the additional chemical modification reduces the gRNA's affinity for the CasX protein and blocks the efficiency of RNP formation. Application of the v5 or v6 profiles yielded competent fractions comparable to, but slightly lower than, those obtained for reactions using the v1 and v2 modifications. While the _kcleavage values between v5 and v6 gRNAs were relatively consistent, the _kcleavage values for both v5 and v6 gRNAs were almost half of those for v1 and v2 gRNAs. The reduced _kcleavage value for v6 gRNA was particularly unexpected given the lack of expected interaction between the gRNA and the CasX protein in the modified elongated stem. However, for both v5 and v6 gRNAs, the reduced gRNA flexibility caused by the 2'OMe modification may inhibit RNP structural changes required for efficient cleavage, or the inclusion of the 2'OMe group may have a negative impact on the modified initial base pair of the hairpin involved in the interaction with the CasX protein. Table 27 : Parameters of cleavage activity assessed for CasX RNPs with various PCSK9- targeting gRNAs using scaffold 235 and containing the indicated chemical modification profiles ( indicated by pattern numbers ) gRNA (scaffold variant-spacer, model number) k _cleavage (min ^-1 ) Fraction capacity 235-6.7, v1 0.901 0.398 235-6.8, v1 1.36 0.398 235-6.7, v2 0.454 0.386 235-6.8, v2 2.03 0.361 235-6.7, v3 0 0 235-6.8, v3 0 0 235-6.7, v4 0.434 0.031 235-6.8, v4 0.257 0.005 235-6.7, v5 0.506 0.313 235-6.8, v5 0.680 0.388 235-6.7, v6 0.462 0.346 235-6.8, v6 0.715 0.325

The editing of the chemically modified PCSK9 targeting gRNA based on scaffold 235 was then assessed in a cell-based assay. CasX mRNA and chemically modified PCSK9 targeting gRNA were co-transfected into HepG2 cells using lipofectamine. Editing levels were measured by indel rates at the PCSK9 locus as determined by NGS and secreted PCSK9 levels as determined by ELISA, and the data are shown in Table 28. The data showed that the use of v3 and v4 gRNAs resulted in the lowest level of editing activity at the PCSK9 locus, consistent with the findings from the biochemical in vitro cleavage assay shown in Table 27. At the same time, the use of v5 and v6 gRNAs resulted in slightly lower editing levels measured by indel rates and PCSK9 secretion than those achieved using v1 and v2 gRNAs (Table 28). Specifically, the results showed that the use of v1 and v2 gRNAs with terminal modifications produced approximately 80-85% editing at the PCSK9 locus, indicating that adding chemical modifications to the gRNA ends is sufficient to achieve efficient editing of CasX. Although the data indicate that efficient editing in vitro is produced using v5 and v6 gRNAs, editing was observed to be close to saturation levels using v1 gRNA in this experiment in which a single dose of gRNA was transfected. Therefore, the use of a single dose makes it challenging to clearly assess the effects of chemical modifications on editing under guide-limiting conditions. Therefore, profiles v1 and v5 were selected for further testing because v1 contains the simplest modification profile and v5 is the most heavily modified profile, which showed robust activity in in vitro applications (Tables 27 and 28). Table 28 : Editing levels measured by indel rates at the PCSK9 locus as determined by NGS and secreted PCSK9 levels as determined by ELISA in HepG2 cells co-transfected with CasX 491 mRNA and various chemically modified PCSK9 targeting gRNAs using scaffold 235 and spacer 6.7 or 6.8 Experimental conditions Insertion/ deletion rate ( editing score) Secreted PCSK9 (ng/mL) average value Standard Deviation average value Standard Deviation CasX mRNA only 0.0021 0.003 52 14 235-6.7, v1 0.83 0.0058 18 5.7 235-6.7, v2 0.79 0.0071 twenty one 4 235-6.7, v3 0.024 0.02 48 19 235-6.7, v4 0.12 0.006 34 5.5 235-6.7, v5 0.73 0.023 twenty one 9 235-6.7, v6 0.75 0.0069 twenty two 8.8 235-6.8, v1 0.85 0.017 16 4.4 235-6.8, v2 0.83 0.0028 20 1.5 235-6.8, v3 0.023 0.0027 39 2.7 235-6.8, v4 0.088 0.0086 42 10 235-6.8, v5 0.77 0.017 19 1.6 235-6.8, v6 0.78 0.014 twenty four 6.9 Non-targeted controls 0.0019 0.0026 42 12

The v1 and v5 profiles were further tested in another cell-based assay to assess their impact on editing efficiency. LNPs were formulated to co-encapsulate CasX 676 mRNA #2 and v1 and v5 chemically modified ROSA26- targeting gRNAs using the newly designed gRNA scaffold 316 (described further in the following subsection). The "v5" profile was slightly modified for application to the 316 scaffold. Three 2'OMe modifications were removed in the region of non-basic pairing close to the 5' of the extended stem to limit modifications to the two stem loop regions. Hepa1-6 hepatocytes were treated with various doses of the resulting LNPs and collected eight days after treatment to assess editing at the ROSA26 locus , which was measured as the insertion/deletion rate detected by NGS (Figure 19). The data indicate that treatment with LNPs delivering the v5 version of the ROSA26 -targeting gRNA resulted in significantly lower levels of editing across the dose range compared to the levels achieved with the v1 counterpart (Figure 19). There are several possible explanations for the difference in relative activity observed using the v5 gRNA in Figure 19 compared to that observed in Table 28. The first and most likely explanation is that the single dose used to achieve editing shown in Table 28 was too high to accurately measure the difference in activity between using the v5 gRNA and the v1 gRNA. It is also possible that the stem loop motif in the 316 version of v5 is removed in vitro, adversely affecting the guided activity. Although it is possible that these modifications provide stability benefits that outweigh the activity costs conferred by the stem-loop modifications, this seems unlikely given that increasing levels of modification have so far resulted in decreased activity. A final possible explanation is that modifications in the v5 profile may negatively affect LNP formulation or behavior through differential interactions between the modified nucleotide backbone and the ionizable lipids of the LNP, potentially leading to less efficient gRNA encapsulation or less efficient gRNA release after internalization.

LNPs co-encapsulating CasX mRNA #2 and chemically modified ROSA26- targeting gRNAs of type v1 and v5 based on scaffold 316 were further tested in vivo. Figure 20 shows the results of the editing analysis, expressed as the percentage of editing measured by the insertion/deletion rate at the ROSA26 locus. The data indicate that under more relevant testing conditions of LNP delivery in vivo, edits achieved using v5 gRNA were reduced by approximately 5-fold compared to edits achieved using v1 gRNA. These findings support the reduced cleavage rate observed biochemically for v5 gRNA in Table 27, indicating that v5 modification has interfered with some aspects of CasX activity. Given the consistent reduction in activity detected in v5 and v6 profiles (Table 27), the reduction in editing can be attributed to modifications in the extended stem region. Although the extension stem of the gRNA has minimal interaction with the CasX protein, the addition of a 2'OMe group at the first base pair has the potential to disrupt the CasX protein-gRNA interaction or the complex RNA folding where the extension stem meets the pseudoknot and triple helix regions. More specifically, the inclusion of a 2'OMe group may adversely affect the base pairing of the gRNA extension stem and residues R49, K50, and K51 of the CasX protein. Finally, structural studies of CasX suggest that gRNA flexibility is required for efficient DNA cleavage (Liu J, et al., CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566:218-223 (2019); Tsuchida CA, et al., Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity. Mol Cell 82(6): 1199-1209 (2022)). Therefore, the addition of 2'OMe groups throughout the extension stem may enforce a more rigid A-form helical structure and prevent gRNA flexibility required for efficient cleavage. In addition, it is possible that additional modifications in the scaffold stem in the v5 and v6 profiles may be detrimental to activity, but given the limited comparison between the v5 and v6 profiles, this is not currently clear.

Design of additional modification profiles aimed at enhancing gRNA stability while mitigating adverse effects on RNP cleavage activity. Using the recently published wild-type CasX structure of Planctomycetes (PDB codes 7WAY, 7WAZ, 7WB0, 7WB1), which has higher homology to the engineered CasX variants evaluated, additional chemical modification profiles of gRNAs were designed and are shown in Figure 12. These profiles show the addition of 2'OMe groups and phosphorothioate bonds in the newly designed gRNA scaffold variants, which are described in the following subsections. These new gRNA chemical modification profiles were designed based on the initial data observed in Table 28 using the v5 gRNA that showed sufficient editing activity, which indicated that modifications to the extension stem and scaffold stem regions did not adversely affect activity. The v7 profile was designed to include 2'OMe at potentially modifiable residues throughout the gRNA structure, but the triple helical region was excluded in view of the significant negative effects previously observed in the v3 profile on the addition of such modifications. The more conservative profiles v8 and v9 were also designed, as shown in Figure 12. For the v8 construct, modifications to the pseudoknot and triple helical loop regions were removed, but modifications to the scaffold stem, extension stem and its flanking single-stranded regions, as well as the 5' and 3' ends were retained. For the v9 profile, modifications of the single-stranded regions flanking the stem loop were removed, but modifications of the stem loop itself as well as the pseudoknot, triple helix loop, and 5' and 3' ends were retained. Additional chemical modification profiles v7, v8, and v9 (discussed further below) of the newly designed gRNA scaffold variant 316 were evaluated in vivo at the PCSK9 locus. The results of the in vivo editing analysis are shown in FIG21 , which are quantitatively measured as the percentage of editing measured by the insertion/deletion rate detected by NGS at the PCSK9 locus. Although low editing efficiencies were detected overall, the data showed that the use of v7, v8, and v9 gRNAs produced lower editing levels at the PCSK9 locus compared to the insertion/deletion rate achieved using v1 gRNA ( FIG21 ). Without wishing to be bound by theory, it is not surprising that the v7, v8, and v9 profiles similarly exhibit relatively low editing activity, given the findings in Figures 19-20 showing low editing activity achieved with the v5 gRNA. As shown in Figure 12, the v7, v8, and v9 profiles include modifications throughout the extended stem region that may interfere with RNP activity. Comparison of gRNA scaffold variants 174 and 316 using in vitro cleavage assays :

Previous work has identified gRNA scaffold variant 235 as the best performing scaffold variant under a variety of delivery conditions. However, the longer length of scaffold 235 (119 bp when using a 20 bp spacer) relative to gRNAs including scaffold 174 (109 bp when using a 20 bp spacer) increases the difficulty of solid-phase RNA synthesis, which will lead to increased manufacturing costs, reduced purity and yield, and higher synthesis failure rates. To address these issues while retaining the improved activity of using scaffold variant 235, a chimeric gRNA scaffold was designed based primarily on the scaffold 235 sequence, but the extended stem loop of scaffold 235 was replaced with the shorter extended stem loop of scaffold variant 174 (Figures 7 to 9). The resulting chimeric scaffold was named scaffold 316, which was synthesized in parallel with scaffold 174 and spacers 6.7 and 6.8 targeting PCSK9 and spacer 7.9 targeting B2M , and had a v1 chemical modification profile with 2'OMe and phosphorothioate bonds on the first and last three nucleotides of all gRNAs (see Table 23). Scaffold 174 was chosen as a comparator rather than scaffold 235 because it was the best characterized scaffold previously and had the same length as variant 316.

The in vitro cleavage activity of gRNAs with scaffolds 174 and 316 and spacers 6.7 and 6.8 was assessed. Cleavage assays were performed with a 20-fold excess of RNP relative to the matched dsDNA target. The cleavage rates of all four guides were quantified and the results are shown in Table 29. The data show that in the case of spacer 6.7, the use of scaffolds 174 or 316 resulted in similar cleavage rates, with cleavage caused by scaffold 316 being slightly faster than that achieved by scaffold 174. In the case of spacer 6.8, the difference in cleavage activity was more pronounced: CasX RNPs using scaffold 316 cleaved DNA almost twice as much as CasX RNPs using scaffold 174 (Table 29).

The analysis was also performed over a longer time course with equimolar amounts of RNP and DNA target to assess the fraction of RNPs expected to have cleavage activity. Because CasX RNPs are essentially single-turnover in the time scale tested, and the concentrations used are expected to be substantially higher than the _KD of the DNA-binding reaction, the amount of cleaved DNA should approximate the amount of active RNP. For spacers 6.7 or 6.8, the active fraction of CasX RNPs incorporating scaffold 316 was 25 to 30% higher than the active fraction of CasX RNPs using scaffold 174 (Table 29). These data suggest that a higher fraction of gRNAs using scaffold 316 are properly folded to bind to the CasX protein, or that gRNAs using scaffold 316 are able to bind to the CasX protein to a greater extent. Compared to scaffold 174, scaffold 316 carries mutations that are expected to stabilize the pseudoknot and triple helix structures required for correct gRNA folding. Specifically, these motifs are more likely to fold incorrectly than simple hairpins found elsewhere in the gRNA structure, so the increased stability of these motifs may result in a slightly higher fraction of gRNAs that fold into the active conformation. Table 29 : Parameters of cleavage activity assessed for CasX RNPs with gRNAs containing scaffold variants 174 or 316 and version 1 (v1) chemical modification profiles . gRNA (Scaffold variant-spacer) K _cracking (min ^-1 ) Fraction capacity 174-6.7, v1 0.236 0.194 174-6.8, v1 0.142 0.165 316-6.7, v1 0.264 0.244 316-6.8, v1 0.272 0.213 Comparison of gRNA scaffold variants 174 and 316 in cell-based assays:

Editing was assessed using gRNA scaffold variant 174 compared to variant 316 in a cell-based assay. CasX 491 mRNA and version 1 (v1) of PCSK9 targeting gRNA using spacers 6.7 and 6.8 were transfected into HepG2 cells via liposomes. Treated cells were collected 28 hours after transfection to analyze editing levels at the PCSK9 locus by NGS and secreted PCSK9 levels by ELISA, and the data are presented in FIG15 . The data show that the use of any of the PCSK9 targeting gRNAs resulted in efficient editing at the PCSK9 locus and significantly reduced PCSK9 secretion compared to the non-targeting control using the B2M targeting gRNA. The results also showed that the use of scaffold 316 produced more efficient editing at the PCSK9 locus than the editing observed using scaffold 174 (approximately 10 percentage point increase in editing rate achieved using scaffold 316 compared to scaffold 174). This finding was further supported by the ELISA results, such that the use of scaffold 316 was more efficient in reducing PCSK9 secretion than the secretion achieved using scaffold 174.

Scaffold variants 174 and 316 were also assessed in an editing analysis, where LNPs were formulated to co-encapsulate CasX 491 mRNA and a B2M- targeting gRNA containing the scaffold variants. HepG2 cells were treated with various doses of the resulting LNPs and harvested seven days after treatment to assess editing at the B2M locus (measured by indel rates detected by NGS ( FIG. 17 )) and loss of B2M-dependent HLA complex surface presentation (detected by flow cytometry ( FIG. 18 )). Results from both analyses showed that treatment with LNPs delivering B2M -targeting gRNAs using scaffold 316 resulted in higher editing efficacy at the B2M locus than LNPs delivering gRNAs using scaffold 174 at all doses ( FIGS. 17 and 18 ). Specifically, at the highest dose of 250 ng, the level of editing produced using scaffold 316 was nearly two-fold higher than that obtained using scaffold 174. This substantial increase in editing efficacy when using scaffold 316 versus scaffold 174 can be attributed to the instability of gRNA structure and folding during LNP formulation, compared to the relative modest differences in activity observed from in vitro cleavage assays. Low pH conditions and the incorporation of cationic lipids during LNP formulation can adversely affect portions of the gRNA structure and cause unfolding. Therefore, it is necessary for the gRNA to rapidly refold in the cytoplasm after delivery in order to bind to the CasX protein to form RNPs and avoid RNase degradation. Mutations in scaffold 316 that increase stability compared to scaffold 174 may provide substantial benefits in supporting proper refolding of the gRNA in the cytoplasm after LNP delivery, while intentional folding protocols for the gRNA prior to biochemical experiments may reduce the impact of these mutations. Example 4 : Design and assessment of editing efficiency of codon-optimized CasX mRNA when delivered in vitro with targeting gRNA

The mRNA sequence and associated modifications can have a significant impact on the efficacy of mRNA-based delivery. Modified nucleotides, including those encoding the 5' cap structure, are important determinants of mRNA stability, translatability, and immunogenicity. Here, a "cap 1" structure was used for all designed and tested CasX mRNAs, which includes 5' m7G, linked to a start nucleotide with a 2'OMe modification via a 5'-5' triphosphate bond. This structure is similar to the "cap 0" structure lacking the 2'OMe modification, which promotes efficient translation and has reduced immunogenicity compared to the "cap 0" structure. In addition, the use of modified nucleobases is expected to reduce the immunogenicity of the mRNA. Here, N1-methyl-pseudouridine was used to replace the uridine riboside for all in vitro transcription reactions because published studies have shown that N1-methyl-pseudouridine substantially enhances mRNA potency and reduces mRNA immunogenicity. The modification is expected to potentially reduce in vivo immunogenicity and increase translation rate by avoiding activation of RIG-I, a major cytosolic sensor of double-stranded RNA, a common contaminant of in vitro transcribed mRNA.

Optimization of the poly(A) tail will also be studied. The poly(A) tail is essential for translation and mRNA stability, with longer tails being associated with longer mRNA half-life. Polyadenylation can be performed transcriptionally with poly(A) polymerase, but this produces variable tail lengths and adds steps to the mRNA production process. Since constructs with plasmids containing 120A tail templates are unstable when propagated in E. coli, often resulting in strains with significantly reduced tail lengths, mRNA production is performed using plasmids containing 80A tail templates and terminated with a type IIS restriction site for flow transcription. An alternative plasmid with a SphI restriction site between the two 60A stretches was also cloned, as published studies have demonstrated that similar constructs are more stable during subcloning and expansion in E. coli and produce mRNA with equivalent activity in mammalian cells. The activity of these alternative forms will be compared across a range of CasX mRNA and gRNA doses using in vitro editing assays to determine the subsequent effects on editing activity. The sequences of the poly(A) tails described herein are listed in Table 30.

The sequences encoding the 5' and 3' UTRs and the codons used for the CasX protein coding sequence contribute to the efficiency of translation. The UTRs were selected from annotated human gene transcripts based on genes previously characterized as having high mRNA stability (e.g., genes encoding α-globin, β-globin), and genes expected or previously shown to be particularly well expressed in the liver (i.e., genes encoding the following proteins: albumin, complement 3, and cytochrome P450 2E1). The sequences of the 5' and 3' UTRs from these various genes are listed in Table 30. For the 3' UTR, the concatenation of individual 3' UTRs was also tested. These constructs were cloned into plasmids containing the T7 promoter, CasX variants 515 or 676, and a poly(A) tail. To separate the effects of individual 5' and 3' UTRs, each UTR was cloned into a construct containing either the 3' or 5' α-globin UTR. IVT was performed and purified by binding to poly(dT) beads to capture the full-length transcript. The resulting mRNA will initially be assessed by co-transfection into HepG2 cells with B2M -targeting gRNA using a range of doses. Editing efficiency will be determined by HLA-immunostaining and flow cytometry. Six days after transfection, cells were harvested for immunostaining of B2M-dependent HLA proteins followed by flow cytometry analysis of B2M protein expression using an Attune ^™ NxT flow cytometer. The best performing individual UTRs will be combined into various configurations, formulated into LNPs, and tested in primary human hepatocytes and mice.

Alternative codon optimizations are also being explored. In addition to CasX codon optimization for other delivery modalities, novel modalities are being designed by constructing a codon usage table based on the usage of ribosomal protein codons and rebalancing the CasX codon usage to match. In addition to potentially improving translation rates, this also effectively causes depletion of uracil bases, which can reduce immunogenicity. This codon optimization is also used to generate mRNA. Additional codon usage has been designed using various available codon optimization tools, adjusting the settings as necessary to achieve a range of GC content levels. These codon optimizations will be tested under similar experimental designs used to test the UTRs described above, and the lead codon optimized CasX candidates will be combined with the lead UTR candidates to generate new CasX leaders for further validation. Table 30 : List of DNA sequences encoding specified elements for the generation and optimization of CasX mRNA describe Coding sequence SEQ ID NO: Poly(A) tail A ₈₀ AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3057 A ₁₂₀ AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3198 A ₆₀ SphIA ₆₀ AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3199 5' UTR α-globulin ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCC 3200 β-Globulin ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACA 3201 albumin CTAGCTTTTCTCTTCTGTCAACCCCACACGCCTTTT 3202 Cytochrome P450 2E1 (CYP2E1) CTCCCGGGCTGGCAGCAGGGCCCCAGC 3203 Complement 3 (C3) ACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCC 3204 3' UTR α-globulin GCTGGAGCCTCGGTGGCCATGCTTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA 3189 albumin CATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAAAGCTTATTCATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAATCTAATAGAGTGGTACAGCACTGT TATTTTTCAAAGATGTGTTGCTATCCTGAAAATTCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCTTATTCCACTTCGGTAGAGGATTTCTAGTTTCTTGTGGGCTAATTAAATAAATCATTAATACTCTTCTAAGTTATGGATTATAAAACATTCAAAATAATATTTTGACATTATGATAATTCTGAATAAAAGAACAAAAACCA 3205 Albumin (truncated) GCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTAGATCT 3206 β-Globulin GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAA 3207 α-globulin + β-globulin GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAGCTCTCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAA 3208 β-globulin + α-globulin GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA 3209 Example 5 : LTRP delivery of LTRP mRNA and targeting gRNA via LNP to achieve inhibition of target loci in vitro

Experiments were performed to assess whether lipid nanoparticles (LNPs) delivering encapsulated LTRP mRNA and targeting gRNA in cell-based assays would induce persistent repression of the target endogenous locus. Materials and Methods: Generation of LTRP mRNA:

mRNA encoding LTRP molecules was produced by IVT as previously described in Example 1. Sequences encoding LTRP molecules were codon optimized as briefly described in Example 1. Examples of DNA sequences encoding LTRP mRNAs are listed in Tables 12 and 31, with corresponding mRNA sequences listed in Tables 13 and 32.

Targeting gRNA (e.g., targeting the endogenous B2M locus) was synthesized as described above in Example 2. LNP formulation:

To formulate LNPs, equal mass ratios of XR or LTRP mRNA and gRNA were diluted in PNI formulation buffer (pH 4.0). GenVoy-ILM ^™ was diluted 1:1 in absolute ethanol. mRNA/gRNA co-formulations were generated using a predetermined N/P ratio. RNA and lipids were passed through a PNI laminar flow filter cartridge on a PNI Ignite™ Benchtop system at a predetermined flow rate ratio. After formulation, LNPs were diluted in PBS (pH 7.4) to reduce ethanol concentration and increase pH, thereby increasing particle stability. Buffer exchange of mRNA/sgRNA-LNPs was achieved by overnight dialysis into PBS (pH 7.4) at 4°C using a 10k Slide-A-Lyzer™ Dialysis Cassette (Thermo Scientific™). After dialysis, mRNA/gRNA-LNPs were concentrated to >0.5 mg/mL using a 100 kDa Amicon®-Ultra centrifugal filter (Millipore) and then filter-sterilized. Formulated LNPs were analyzed on a Stunner (Unchained Labs) to determine their diameter and polydispersity index (PDI). Encapsulation efficiency and RNA concentration were determined by RiboGreen™ analysis using Invitrogen's Quant-iT™ RiboGreen™ RNA Assay Kit. LNPs encapsulating LTRP mRNA and targeting gRNA were delivered to mouse liver Hepa1-6 cells:

Hepa1-6 cells were seeded in 96-well plates. The next day, the seeded cells were treated with different concentrations of LNPs, which were prepared in six 2-fold serial dilutions starting at 250 ng. These LNPs were formulated to encapsulate LTRP mRNA and B2M targeting gRNA. The medium was changed 24 hours after LNP treatment, and the cells were cultured and then collected at multiple time points (e.g., 7 days, 14 days, 21 days, 28 days, and 56 days after treatment) for gDNA extraction for editing assessment of the B2M locus by NGS and bisulfite sequencing to assess off-target methylation at the VEGFA locus. Bisulfite sequencing to assess LTRP specificity as measured by off-target methylation levels at the target locus:

To determine the off-target methylation level at the B2M locus, gDNA was extracted from the collected cells using the Zymo Quick-DNA Miniprep Plus kit according to the manufacturer's instructions. The extracted gDNA was then bisulfite converted using the EZ DNA Methylation ^TM kit (Zymo) according to the manufacturer's protocol to convert all non-methylated cytosines to uracil. The resulting bisulfite-treated DNA was then sequenced using next-generation sequencing (NGS) to determine the off-target methylation level at the B2M and VEGFA loci. NGS processing and analysis:

Target amplicon was amplified by PCR from 100 ng of bisulfite treated DNA using a set of primers specific for the bisulfite converted target loci of interest (human B2M and VEGFA loci). These gene specific primers contain additional sequence at the 5′ end to introduce the Illumina ^™ adapter. Amplified DNA products were purified using the Cytiva Sera-Mag Select DNA Cleanup Kit. Amplicon quality and quantification were assessed using the Fragment Analyzer DNA Analysis Kit (Agilent, dsDNA 35-1500 bp). Amplicon was sequenced on an Illumina ^™ Miseq ^™ according to the manufacturer's instructions. The sequenced raw fastq files were processed using the Bismark Bisulfite Read Mapper and Methylation caller. PCR amplification of bisulfite treated DNA will convert all uracil nucleotides to thymine, and sequencing of the PCR products will determine the cytosine to thymine conversion rate as a readout of potential off-target methylation levels at the B2M and VEGFA loci mediated by each LTRP molecule.

It is expected that the results of this experiment show that LTRP mRNA and targeting gRNA can be co-encapsulated in LNPs to be delivered to target cells to induce transmissible transcription of target endogenous loci. Table 31 : Coding sequences of LTRP1-ZIM3 and LTRP5-ZIM3 mRNA molecules evaluated in Experiment #2 of this Example * LTRP ID Components DNA Sequence SEQ ID NO: LTRP1-ZIM3-KRAB 5'UTR GACCGGCCGCCACC 3082 Start codon + NLS + linker ATGGCCCCAAAGAAGAAGCGGAAGGTCTCTAGAGTTAACGGATCAGGCTCTGGAGGTGGA 3083 Start codon + DNMT3A catalytic domain ATGAACCATGACCAGGAATTTGACCCCCCAAAGGTTTACCCACCTGTGCCAGCTGAGAAGGAAGCCCATCCGCGTGCTGTCTCTCTTGATGGGATTGCTACAGGGCTCCTGGTGCTGAAGGACCTGGGCATCCAAGTGGACCGCTACATCGCCTCCGAGGTGTGTGAGGACTCCATCACGGTGGGCATGGTGCGGCACCAGGGAAAGATCATGTACGTCGGGG ACGTCCGCAGCGTCACACAGAAGCATATCCAGGAGTGGGGCCCATTCGACCTGGTGATTGGAGGCAGTCCCTGCAACGACCTCTCCATTGTCAACCCTGCCCGCAAGGGACTTTATGAGGGTACTGGCCGCCTCTTCTTTGAGTTCTACCGCCTCCTGCATGATGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTTTGAGAATGTGGTGGCCATG GGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTTGAGTCTAACCCCGTGATGATTGACGCCAAAGAAGTGTCTGCTGCACACAGGGCCCGTTACTTCTGGGGTAACCTTCCTGGCATGAACAGGCCTTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAAGAGTGTCTGGAGCACGGCAGAATAGCCAAGTTCAGCAAAGTGAGGACCATTACCACCAGGT CAAACTCTATAAAGCAGGGCAAAGACCAGCATTTCCCCGTCTTCATGAACGAAGGAGGACATCCTGTGGTGCACTGAAATGGAAAGGGTGTTTGGCTTCCCCGTCCACTACACAGACGTGTCCAACATGAGCCGCTTGGCGAGGCAGAGACTGCTGGGCCGGTCGTGGAGCGTGCCGGTCATCCGCCACCTCTTCGCTCCGCTGAAGGAATATTTTGCTTGTGTG 3084 Connector TCTAGCGGCAATAGTAACGCTAACAGCCGCGGGCCGAGCTTCAGCAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAGCCAT 3085 DNMT3L interaction domain ATGGGCCCTATGGAGATATACAAGACAGTGTCTGCATGGAAGAGACAGCCAGTGCGGGTACTGAGCCTCTTCAGAAACATCGACAAGGTACTAAAGAGTTTGGGCTTCTTGGAAAGCGGTTCTGGTTCTGGGGGAGGAACGCTGAAGTACGTGGAAGATGT CACAAATGTCGTGAGGAGGGACGTGGAGAAATGGGGCCCCTTTGACCTGGTGTACGGCTCGACGCAGCCCCTAGGCAGCTCTTGTGATCGCTGTCCCGGCTGGTACATGTTCCAGTTCCACCGGATCCTGCAGTATGCGCTGCCTCGCCAGGAGAGTCAGC GGCCCTTCTTCTGGATATTCATGGACAATCTGCTGCTGACTGAGGATGACCAAGAGACAACTACCCGCTTCCTTCAGACAGAGGCTGTGACCCTCCAGGATGTCCGTGGCAGAGACTACCAGAATGCTATGCGGGTGTGGAGCAACATTCCAGGGCTGAAG AGCAAGCATGCGCCCCTGACCCCAAAGGAAGAAGAGTATCTGCAAGCCCAAGTCAGAAGCAGGAGCAAGCTGGACGCCCCGAAAGTTGACCTCCTGGTGAAGAACTGCCTTCTCCCGCTGAGAGAGTACTTCAAGTATTTTTCTCAAAACTCACTTCCTCTT 3086 Connector GGAGGGCCGAGCTCTGGCGCACCCCCACCAAGTGGAGGGTCTCCTGCCGGGTCCCCCAACATCTACTGAAGAAGGCACCAGCGAATCCGCAACGCCCGAGTCAGGCCCTGGTACCTCCACAGAACCATCTGAAGGTAGTGCGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCACCGAAGAAGGCACGTCAACCGAACCAAGTGAAGGATCTGCCCCTGGGACCAGCACTGAACCATCTGAG 3087 Connector + Buffer GGCGGTTCCGGCGGAGGAAGCGCT 3088 dCasX491 CAAGAGATCAAGAGAATCAACAAGATCAGAAGGAGACTGGTCAAGGACAGCAACACAAAGAAGGCCGGCAAGACAGGCCCCATGAAAACCCTGCTCGTCAGAGTGATGACCCCTGACCTGAGAGAGCGGCTGGAAAACCTGAGAAAGAAGCCCGAGAACATCCCTCAGCCTATCAGCAACACC AGCAGGGCCAACCTGAACAAGCTGCTGACCGACTACACCGAGATGAAGAAAGCCATCCTGCACGTGTACTGGGAAGAGTTCCAGAAAGACCCCGTGGGCCTGATGAGCAGAGTTGCTCAGCCTGCCAGCAAGAAGATCGACCAGAACAAGCTGAAGCCCGAGATGGACGAGAAGGGCAATCTG ACCACAGCCGGCTTTGCCTGCTCTCAGTGTGGCCAGCCTCTGTTCGTGTACAAGCTGGAACAGGTGTCCGAGAAAGGCAAGGCCTACACCAACTTCGGCAGATGTAACGTGGCCGAGCACGAGAAGCTGATTCTGCTGGCCCAGCTGAAACCTGAGAAGGACTCTGATGAGGCCGTGACC TACAGCCTGGGCAAGTTTGGACAGAGAGCCCTGGACTTCTACAGCATCCACGTGACCAAAGAAAGCACACACCCCGTGAAGCCCCTGGCTCAGATCGCCGGCAATAGATACGCCTCTGGACCTGTGGGCAAAGCCCTGTCCGATGCCTGCATGGGAACAATCGCCAGCTTCCTGAGCAAGTAC CAGGACATCATCGAGCACCAGAAGGTGGTCAAGGGCAACCAGAAGAGACTGGAAAGCCTGAGGGAGCTGGCCGGCAAAGAGAACCTGGAATACCCCAGCGTGACCCTGCCTCCTCAGCCTCACACAAAAGAAGGCGTGGACGCCTACAACGAAGTGATCGCCAGAGTGAGAATGTGGGTC AACCTGAACCTGTGGCAGAAGCTGAAACTGTCCAGGGACGACGCCAAGCCTCTGCTGAGACTGAAGGGCTTCCCTAGCTTCCCTCTGGTGGAAAGACAGGCCAATGAAGTGGATTGGTGGGACATGGTCTGCAACGTGAAGAAGCTGATCAACGAGAAGAAAGAGGATGGCAAGGTTTTCTGG CAGAACCTGGCCGGCTACAAGAGACAAGAAGCCCTGAGGCCTTACCTGAGCAGCGAAGAGGACCGGAAGAAGGGCAAGAAGTTCGCCAGATACCAGCTGGGCGACCTGCTGCTGCACCTGGAAAAGAAGCACGGCGAGGACTGGGGCAAAGTGTACGATGAGGCCTGGGAGAGAATCGACAAG AAGGTGGAAGGCCTGAGCAAGCACATTAAGCTGGAAGAGGAAAGAAGGAGCGAGGACGCCCAATCTAAAGCCGCTCTGACCGATTGGCTGAGAGCCAAGGCCAGCTTTGTGATCGAGGGCCTGAAAGAGGCCGACAAGGACGAGTTCTGCAGATGCGAGCTGAAGCTGCAGAAGTGGTACGGCG ATCTGAGAGGCAAGCCCTTCGCCATTGAGGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCAGTACAACTGCGCCTTCATTTGGCAGAAAGACGGCGTCAAGAAACTGAACCTGTACCTGATCATCAATTACTTCAAAGGCGGCAAGCTGCGGTTCAAGAAGATCAAACCCGAGG CCTTCGAGGCTAACAGATTCTACACCGTGATCAACAAAAAGTCCGGCGAGATCGTGCCCATGGAAGTGAACTTCAACTTCGACGACCCCAACCTGATTATCCTGCCTCTGGCCTTCGGCAAGAGACAGGGCAGAGAGTTCATCTGGAACGATCTGCTGAGCCTGGAAACCGGCTCTCTGAAGC TGGCCAATGGCAGAGTGATCGAGAAAACCCTGTACAACAGGAGAACCAGACAGGACGAGCCTGCTCTGTTTGTGGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGCAACATCAAGCCCATGAACCTGATCGGCGTGGCCCGGGGCGAGAATATCCCTGCTGTGATCGCCCTGACAG ACCCTGAAGGATGCCCACTGAGCAGATTCAAGGACTCCCTGGGCAACCCTACACACATCCTGAGAATCGGCGAGAGCTACAAAGAGAAGCAGAGGACAATCCAGGCCAAGAAAGAGGTGGAACAGAGAAGAGCCGGCGGATACTCTAGGAAGTACGCCAGCAAGGCCAAGAATCTGGCCGACGA CATGGTCCGAAACACCGCCAGAGATCTGCTGTACTACGCCGTGACACAGGACGCCATGCTGATCTTCGCGAATCTGAGCAGAGGCTTCGGCCGGCAGGGCAAGAGAACCTTTATGGCCGAGAGGCAGTACACCAGAATGGAAGATTGGCTCACAGCTAAACTGGCCTACGAGGGACTGAGCAA GACCTACCTGTCCAAAACACTGGCCCAGTATACCTCCAAGACCTGCAGCAATTGCGGCTTCACCATCACCAGCGCCGACTACGACAGAGTGCTGGAAAAGCTCAAGAAAACCGCCACCGGCTGGATGACCACCATCAACGGCAAAGAGCTGAAGGTTGAGGGCCAGATCACCTACTACAACAG GTACAAGAGGCAGAACGTCGTGAAGGATCTGAGCGTGGAACTGGACAGACTGAGCGAAGAGAGCGTGAACAACGACATCAGCAGCTGGACAAAGGGCAGATCAGGCGAGGCTCTGAGCCTGCTGAAGAAGAGGTTTAGCCACAGACCTGTGCAAGAGAAGTTCGTGTGCCTGAACTGCGGCTT CGAGACACACGCCGCTGAACAGGCTGCCCTGAACATTGCCAGAAGCTGGCTGTTCCTGAGAAGCCAAGAGTACAAGAAGTACCAGACCAACAAGACCACCGGCAACACCGACAAGAGGGCCTTTGTGGAAAACCTGGCAGAGCTTCTACAGAAAAAAGCTGAAAGAAGTCTGGAAGCCCGCCGTG 3089 Connector + Buffer CGATCGGGCGGTTCCGGCGGAGGTTCCACTAGT 3090 ZIM3-KRAB ATGAACAATTCCCAGGGAAGAGTGACCTTCGAGGATGTCACTGTGAACTTCACCCAGGGGGAGTGGCAGCGGCTGAATCCCGAACAGAGAAACTTGTACAGGGATGTGATGCTGGAGAATTACAGCAACCTTGTCTCTGTGGGACAAGGG GAAACCACCAAACCCGATGTGATCTTGAGGTTGGAACAAGGAAAGGAGCCATGGTTGGAGGAAGAGGAAGTGCTGGGAAGTGGCCGTGCAGAAAAAAATGGGGACATTGGAGGGCAGATTTGGAAGCCAAAGGATGTGAAAGAGAGTCTC 3091 Buffer + NLS ACTAGTCCAAAAAAGAAGAGAAAGGTA 3092 Logo GATTACAAAGATGACGATGACAAAGACTACAAGGATGATGATGATAAG 3093 Buffer GGATCCGGCTG 3094 Poly(A) tail AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3095 LTRP5-ZIM3-KRAB 5'UTR GACCGGCCGCCACC 3082 Start codon + NLS + buffer ATGGCCCCAAAGAAGAAGCGGAAGGTCTCTAGA 3096 Start codon + DNMT3A catalytic domain ATGAACCATGACCAGGAATTTGACCCCCCAAAGGTTTACCCACCTGTGCCAGCTGAGAAGGAAGCCCATCCGCGTGCTGTCTCTCTTGATGGGATTGCTACAGGGCTCCTGGTGCTGAAGGACCTGGGCATCCAAGTGGACCGCTACATCGCCTCCGAGGTGTGTGAGGACTCCATCACGGTGGGCATGGTGCGGCACCAGGGAAAGATCATGTACGTCGGGG ACGTCCGCAGCGTCACACAGAAGCATATCCAGGAGTGGGGCCCATTCGACCTGGTGATTGGAGGCAGTCCCTGCAACGACCTCTCCATTGTCAACCCTGCCCGCAAGGGACTTTATGAGGGTACTGGCCGCCTCTTCTTTGAGTTCTACCGCCTCCTGCATGATGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTTTGAGAATGTGGTGGCCATG GGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTTGAGTCTAACCCCGTGATGATTGACGCCAAAGAAGTGTCTGCTGCACACAGGGCCCGTTACTTCTGGGGTAACCTTCCTGGCATGAACAGGCCTTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAAGAGTGTCTGGAGCACGGCAGAATAGCCAAGTTCAGCAAAGTGAGGACCATTACCACCAGGT CAAACTCTATAAAGCAGGGCAAAGACCAGCATTTCCCCGTCTTCATGAACGAAGGAGGACATCCTGTGGTGCACTGAAATGGAAAGGGTGTTTGGCTTCCCCGTCCACTACACAGACGTGTCCAACATGAGCCGCTTGGCGAGGCAGAGACTGCTGGGCCGGTCGTGGAGCGTGCCGGTCATCCGCCACCTCTTCGCTCCGCTGAAGGAATATTTTGCTTGTGTG 3084 Connector TCTAGCGGCAATAGTAACGCTAACAGCCGCGGGCCGAGCTTCAGCAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAGCCAT 3085 DNMT3L interaction domain ATGGGCCCTATGGAGATATACAAGACAGTGTCTGCATGGAAGAGACAGCCAGTGCGGGTACTGAGCCTCTTCAGAAACATCGACAAGGTACTAAAGAGTTTGGGCTTCTTGGAAAGCGGTTCTGGTTCTGGGGGAGGAACGCTGAAGTACGTGGAAGATGT CACAAATGTCGTGAGGAGGGACGTGGAGAAATGGGGCCCCTTTGACCTGGTGTACGGCTCGACGCAGCCCCTAGGCAGCTCTTGTGATCGCTGTCCCGGCTGGTACATGTTCCAGTTCCACCGGATCCTGCAGTATGCGCTGCCTCGCCAGGAGAGTCAGC GGCCCTTCTTCTGGATATTCATGGACAATCTGCTGCTGACTGAGGATGACCAAGAGACAACTACCCGCTTCCTTCAGACAGAGGCTGTGACCCTCCAGGATGTCCGTGGCAGAGACTACCAGAATGCTATGCGGGTGTGGAGCAACATTCCAGGGCTGAAG AGCAAGCATGCGCCCCTGACCCCAAAGGAAGAAGAGTATCTGCAAGCCCAAGTCAGAAGCAGGAGCAAGCTGGACGCCCCGAAAGTTGACCTCCTGGTGAAGAACTGCCTTCTCCCGCTGAGAGAGTACTTCAAGTATTTTTCTCAAAACTCACTTCCTCTT 3086 Connector GGCGGTTCCGGCGGAGGA 3097 ZIM3-KRAB ATGAACAATTCCCAGGGAAGAGTGACCTTCGAGGATGTCACTGTGAACTTCACCCAGGGGGAGTGGCAGCGGCTGAATCCCGAACAGAGAAACTTGTACAGGGATGTGATGCTGGAGAATTACAGCAACCTTGTCTCTGTGGGACAAGGG GAAACCACCAAACCCGATGTGATCTTGAGGTTGGAACAAGGAAAGGAGCCATGGTTGGAGGAAGAGGAAGTGCTGGGAAGTGGCCGTGCAGAAAAAAATGGGGACATTGGAGGGCAGATTTGGAAGCCAAAGGATGTGAAAGAGAGTCTC 3091 Connector GGAGGGCCGAGCTCTGGCGCACCCCCACCAAGTGGAGGGTCTCCTGCCGGGTCCCCCAACATCTACTGAAGAAGGCACCAGCGAATCCGCAACGCCCGAGTCAGGCCCTGGTACCTCCACAGAACCATCTGAAGGTAGTGCGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCACCGAAGAAGGCACGTCAACCGAACCAAGTGAAGGATCTGCCCCTGGGACCAGCACTGAACCATCTGAG 3087 dCasX491 CAAGAGATCAAGAGAATCAACAAGATCAGAAGGAGACTGGTCAAGGACAGCAACACAAAGAAGGCCGGCAAGACAGGCCCCATGAAAACCCTGCTCGTCAGAGTGATGACCCCTGACCTGAGAGAGCGGCTGGAAAACCTGAGAAAGAAGCCCGAGAACATCCCTCAGCCTATCAGCAACACC AGCAGGGCCAACCTGAACAAGCTGCTGACCGACTACACCGAGATGAAGAAAGCCATCCTGCACGTGTACTGGGAAGAGTTCCAGAAAGACCCCGTGGGCCTGATGAGCAGAGTTGCTCAGCCTGCCAGCAAGAAGATCGACCAGAACAAGCTGAAGCCCGAGATGGACGAGAAGGGCAATCTG ACCACAGCCGGCTTTGCCTGCTCTCAGTGTGGCCAGCCTCTGTTCGTGTACAAGCTGGAACAGGTGTCCGAGAAAGGCAAGGCCTACACCAACTTCGGCAGATGTAACGTGGCCGAGCACGAGAAGCTGATTCTGCTGGCCCAGCTGAAACCTGAGAAGGACTCTGATGAGGCCGTGACC TACAGCCTGGGCAAGTTTGGACAGAGAGCCCTGGACTTCTACAGCATCCACGTGACCAAAGAAAGCACACACCCCGTGAAGCCCCTGGCTCAGATCGCCGGCAATAGATACGCCTCTGGACCTGTGGGCAAAGCCCTGTCCGATGCCTGCATGGGAACAATCGCCAGCTTCCTGAGCAAGTAC CAGGACATCATCGAGCACCAGAAGGTGGTCAAGGGCAACCAGAAGAGACTGGAAAGCCTGAGGGAGCTGGCCGGCAAAGAGAACCTGGAATACCCCAGCGTGACCCTGCCTCCTCAGCCTCACACAAAAGAAGGCGTGGACGCCTACAACGAAGTGATCGCCAGAGTGAGAATGTGGGTC AACCTGAACCTGTGGCAGAAGCTGAAACTGTCCAGGGACGACGCCAAGCCTCTGCTGAGACTGAAGGGCTTCCCTAGCTTCCCTCTGGTGGAAAGACAGGCCAATGAAGTGGATTGGTGGGACATGGTCTGCAACGTGAAGAAGCTGATCAACGAGAAGAAAGAGGATGGCAAGGTTTTCTGG CAGAACCTGGCCGGCTACAAGAGACAAGAAGCCCTGAGGCCTTACCTGAGCAGCGAAGAGGACCGGAAGAAGGGCAAGAAGTTCGCCAGATACCAGCTGGGCGACCTGCTGCTGCACCTGGAAAAGAAGCACGGCGAGGACTGGGGCAAAGTGTACGATGAGGCCTGGGAGAGAATCGACAAG AAGGTGGAAGGCCTGAGCAAGCACATTAAGCTGGAAGAGGAAAGAAGGAGCGAGGACGCCCAATCTAAAGCCGCTCTGACCGATTGGCTGAGAGCCAAGGCCAGCTTTGTGATCGAGGGCCTGAAAGAGGCCGACAAGGACGAGTTCTGCAGATGCGAGCTGAAGCTGCAGAAGTGGTACGGCG ATCTGAGAGGCAAGCCCTTCGCCATTGAGGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCAGTACAACTGCGCCTTCATTTGGCAGAAAGACGGCGTCAAGAAACTGAACCTGTACCTGATCATCAATTACTTCAAAGGCGGCAAGCTGCGGTTCAAGAAGATCAAACCCGAGG CCTTCGAGGCTAACAGATTCTACACCGTGATCAACAAAAAGTCCGGCGAGATCGTGCCCATGGAAGTGAACTTCAACTTCGACGACCCCAACCTGATTATCCTGCCTCTGGCCTTCGGCAAGAGACAGGGCAGAGAGTTCATCTGGAACGATCTGCTGAGCCTGGAAACCGGCTCTCTGAAGC TGGCCAATGGCAGAGTGATCGAGAAAACCCTGTACAACAGGAGAACCAGACAGGACGAGCCTGCTCTGTTTGTGGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGCAACATCAAGCCCATGAACCTGATCGGCGTGGCCCGGGGCGAGAATATCCCTGCTGTGATCGCCCTGACAG ACCCTGAAGGATGCCCACTGAGCAGATTCAAGGACTCCCTGGGCAACCCTACACACATCCTGAGAATCGGCGAGAGCTACAAAGAGAAGCAGAGGACAATCCAGGCCAAGAAAGAGGTGGAACAGAGAAGAGCCGGCGGATACTCTAGGAAGTACGCCAGCAAGGCCAAGAATCTGGCCGACGA CATGGTCCGAAACACCGCCAGAGATCTGCTGTACTACGCCGTGACACAGGACGCCATGCTGATCTTCGCGAATCTGAGCAGAGGCTTCGGCCGGCAGGGCAAGAGAACCTTTATGGCCGAGAGGCAGTACACCAGAATGGAAGATTGGCTCACAGCTAAACTGGCCTACGAGGGACTGAGCAA GACCTACCTGTCCAAAACACTGGCCCAGTATACCTCCAAGACCTGCAGCAATTGCGGCTTCACCATCACCAGCGCCGACTACGACAGAGTGCTGGAAAAGCTCAAGAAAACCGCCACCGGCTGGATGACCACCATCAACGGCAAAGAGCTGAAGGTTGAGGGCCAGATCACCTACTACAACAG GTACAAGAGGCAGAACGTCGTGAAGGATCTGAGCGTGGAACTGGACAGACTGAGCGAAGAGAGCGTGAACAACGACATCAGCAGCTGGACAAAGGGCAGATCAGGCGAGGCTCTGAGCCTGCTGAAGAAGAGGTTTAGCCACAGACCTGTGCAAGAGAAGTTCGTGTGCCTGAACTGCGGCTT CGAGACACACGCCGCTGAACAGGCTGCCCTGAACATTGCCAGAAGCTGGCTGTTCCTGAGAAGCCAAGAGTACAAGAAGTACCAGACCAACAAGACCACCGGCAACACCGACAAGAGGGCCTTTGTGGAAAACCTGGCAGAGCTTCTACAGAAAAAAGCTGAAAGAAGTCTGGAAGCCCGCCGTG 3089 Connector + Buffer CGATCGGGCGGTTCCGGCGGAGGTTCCACTAGT 3090 NLS CCAAAAAAGAAGAGAAAGGTA 3098 Logo GATTACAAAGATGACGATGACAAAGACTACAAGGATGATGATGATAAG 3093 Buffer GGATCCGGCTG 3094 Poly(A) tail AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3095 * The components are listed in 5' to 3' order within the construct Table 32 : Full-length RNA sequences of LTRP1-ZIM3 and LTRP5-ZIM3 mRNA molecules evaluated in Experiment #2 of this example . Modification " mψ " = N1- methyl - pseudouridine LTRP ID RNA- seq SEQ ID NO: LTRP1-ZIM3-KRAB GACCGGCCGCCACCAmψGGCCCCAAAGAAGAAGCGGAAGGmψCmψCmψAGAGmψmψAACGGAmψCAGGCmψCmψGGAGGmψGGAAmψGAACCAmψGACC AGGAAmψmψmψGACCCCCCAAAGGmψmψmψACCCACCmψGmψGCCAGCmψGAGAAGAGGAAGCCCAmψCCGCGmψGCmψGmψCmψCmψCmψmψmψGAmψ GGGAmψmψGCmψACAGGGCmψCCmψGGmψGCmψGAAGGACCmψGGGCAmψCCAAGmψGGACCGCmψACAmψCGCCmψCCGAGGmψGmψGmψGAGGACmψ CCAmψCACGGmψGGGCAmψGGmψGCGGCACCAGGGAAAGAmψCAmψGmψACGmψCGGGGACGmψCCGCAGCGmψCACACAGAAGCAmψAmψCCAGGAGmψ GGGGCCCAmψmψCGACCmψGGmψGAmψmψGGAGGCAGmψCCCmψGCAACGACCmψCmψCCAmψmψGmψCAACCCmψGCCCGCAAGGGACmψmψmψAmψG AGGGmψACmψGGCCGCCmψCmψmψCmψmψmψGAGmψmψCmψACCGCCmψCCmψGCAmψGAmψGCGCGGCCCAAGGAGGGAGamψGAmψCGCCCCmψmψC mψmψCmψGGCmψCmψmψmψGAGAAmψGmψGGmψGGCCAmψGGGCGmψmψAGmψGACAAGAGGGACAmψCmψCGCGAmψmψmψCmψmψGAGmψCmψAACC CCGmψGAmψGAmψmψGACGCCAAAGAAGmψGmψCmψGCmψGCACACAGGGCCCGmψmψACmψmψCmψGGGGmψAACCmψmψCCmψGGCAmψGAACAGGCC mψmψmψGGCAmψCCACmψGmψGAAmψGAmψAAGCmψGGAGCmψGCAAGAGmψGmψCmψGGAGCACGGCAGAAmψAGCCAAGmψmψCAGCAAAGmψGAGG ACCAmψmψACCACCAGGmψCAAACmψCmψAmψAAAGCAGGGCAAAGACCAGCAmψmψmψCCCCGmψCmψmψCAmψGAACGAGAAGGAGGACAmψCCmψG mψGGmψGCACmψGAAAmψGGAAAGGGmψGmψmψmψGGCmψmψCCCCGmψCCACmψACACAGACGmψGmψCCAACAmψGAGCCGCmψmψGGCGAGGCAGA GACmψGCmψGGGCCGGmψCGmψGGAGCGmψGCCGGmψCAmψCCGCCACCmψCmψmψCGCmψCCGCmψGAAGGAAmψAmψmψmψmψmψGCmψmψGmψGmψGmψ CmψAGCGGCAAmψAGmψAACGCmψAACAGCCGCGGGCCGAGCmψmψCAGCAGCGGCCmψGGmψGCCGmψmψAAGCmψmψGCGCGGCAGCCAmψAmψGGG CCCmψAmψGGAGAmψAmψACAAGACAGmψGmψCmψGCAmψGGAAGAGACAGCCAGmψGCGGGmψACmψGAGCCmψCmψmψCAGAAACAmψCGACAAGGmψ ACmψAAAGAGmψmψmψGGGCmψmψCmψmψGGAAAGCGGmψmψCmψGGmψmψCmψGGGGGAGGAACGCmψGAAGmψACGmψGGAAGAmψGmψCACAAAmψ GmψCGmψGAGGAGGGACGmψGGAGAAAmψGGGGCCCCmψmψmψGACCmψGGmψGmψACGGCmψCGACGCAGCCCCmψAGGCAGCmψCmψmψGmψGAmψCG CmψGmψCCCGGCmψGGmψACAmψGmψmψCCAGmψmψCCACCGGAmψCCmψGCAGmψAmψGCGCmψGCCmψCGCCAGGAGAGmψCAGCGGCCCmψmψCmψ mψCmψGGAmψAmψmψCAmψGGACAAmψCmψGCmψGCmψGACmψGAGGAmψGACCAAGAGACAACmψACCCGCmψmψCCmψmψCAGACAGAGGCmψGmψG ACCCmψCCAGGAmψGmψCCGmψGGCAGAGACmψACCAGAAmψGCmψAmψGCGGGmψGmψGGAGCAACAmψmψCCAGGGCmψGAAGAGCAAGCAmψGCGC CCCmψGACCCCAAAGGAAGAAGAGmψAmψCmψGCAAGCCCAAGmψCAGAAGCAGGAGCAAGCmψGGACGCCCGAAAGmψmψGACCmψCCmψGGmψGAAG AACmψGCCmψmψCmψCCCGCmψGAGAGAGmψACmψmψCAAGmψAmψmψmψmψmψCmψCAAAACmψCACmψmψCCmψCmψmψGGAGGGCCGAGCmψCmψG GCGCACCCCCACCAAGmψGGAGGGmψCmψCCmψGCCGGGmψCCCCAACAmψCmψACmψGAAGAAGGCACCAGCGAAmψCCGCAACGCCCGAGmψCAGGC CCmψGGmψACCmψCCACAGAACCAmψCmψGAAGGmψAGmψGCGCCmψGGmψmψCCCCAGCmψGGAAGCCCmψACmψmψCCACCGAAGAAGGCACGmψCA ACCGAACCAAGmψGAAGGAmψCmψGCCCCmψGGGACCAGCACmψGAACCAmψCmψGAGGGCGGmψmψCCGGCGGAGGAAGCGCmψCAAGAGAmψCAAGAG AAmψCAACAAGAmψCAGAAGGAGACmψGGmψCAAGGACAGCAACACAAAGAAGGCCGGCAAGACAGGCCCCAmψGAAAACCCmψGCmψCGmψCAGAGmψGAmψGACCCCmψGACCmψGAGAGAGCGGCmψGGAAAACCmψGAGAAAGAAGCCCGAGAACAmψCCCmψCAGCCmψAmψCAGCAACACCAGCAGGGCCAA CCmψGAACAAGCmψGCmψGACCGACmψACACCGAGAmψGAAGAAAGCCAmψCCmψGCACGmψGmψACmψGGGAAGAGmψmψCCAGAAAGACCCCGmψGG GCCmψGAmψGAGCAGAGmψmψGCmψCAGCCmψGCCAGCAAGAAGAmψCGACCAGAACAAGCmψGAAGCCCGAGAmψGGACGAGAAGGGCAAmψCmψGACC ACAGCCGGCmψmψmψGCCmψGCmψCmψCAGmψGmψGGCCAGCCmψCmψGmψmψCGmψGmψACAAGCmψGGAACAGGmψGmψCCGAGAAAGGCAAGGCCm ψACACCAACmψACmψmψCGGCAGAmψGmψAACGmψGGCCGAGCACGAGAAGCmψGAmψmψCmψGCmψGGCCCAGCmψGAAACCmψGAGAAGGACmψCmψG AmψGAGGCCGmψGACCmψACAGCCmψGGGCAAGmψmψmψGGACAGAGAGCCCmψGGACmψmψCmψACAGCAmψCCACGmψGACCAAAGAAAGCACACAC CCCGmψGAAGCCCCmψGGCmψCAGAmψCGCCGGCAAmψAGAmψACGCCmψCmψGGACCmψGmψGGGCAAAGCCCmψGmψCCGAmψGCCmψGCAmψGGGAA CAAmψCGCCAGCmψmψCCmψGAGCAAGmψACCAGGACAmψCAmψCAmψCGAGCACCAGAAGGmψGGmψCAAGGGCAACCAGAAGAGACmψGGAAAGCCm ψGAGGGAGCmψGGCCGGCAAAGAGAACCmψGGAAmψACCCCAGCGmψGACCCmψGCCmψCCmψCAGCCmψCACACAAAAGAAGGCGmψGGACGCCmψAC AACGAAGmψGAmψCGCCAGAGmψGAGAAmψGmψGGGmψCAACCmψGAACCmψGmψGGCAGAAGCmψGAAACmψGmψCCAGGGACGACGCCAAGCCmψCm ψGCmψGAGACmψGAAGGGCmψmψCCCmψAGCmψmψCCCmψCmψGGmψGGAAAGACAGGCCAAmψGAAGmψGGAmψmψGGmψGGGACAmψGGmψCmψGCAA CGmψGAAGAAGCmψGAmψCAACGAGAAGAAAGAGGAmψGGCAAGGmψmψmψmψCmψGGCAGAACCmψGGCCGGCmψACAAGAGACAAGAAGCCCmψGAGGCCmψmψACCmψGAGCAGCGAAGAGGACCGGAAGAAGGGCAAGAAGmψmψCGCCAGAmψACCAGCmψGGGCGACCmψGCmψGCmψGCACCmψGGAAAAG AAGCACGGCGAGGACmψGGGGCAAAGmψGmψACGAmψGAGGCCmψGGGAGAGAAmψCGACAAGAAGGmψGGAAGGCCmψGAGCAAGCACAmψmψAAGCm ψGGAAGAGGAAAGAAGGAGCGAGGACGCCCAAmψCmψAAAGCCGCmψCmψGACCGAmψmψGGCmψGAGAGCCAAGGCCAGCmψmψmψGmψGAmψCGAGGG CCmψGAAAGAGGCCGACAAGGACGAGmψmψCmψGCAGAmψGCGAGCmψGAAGCmψGCAGAAGmψGGmψACGGCGAmψCmψGAGAGGCAAGCCCmψmψCG CCAmψmψGAGGCCGAGAACAGCAmψCCmψGGACAmψCAGCGGCmψmψCAGCAAGCAGmψACAACmψGCGCCmψmψCAmψmψmψGGCAGAAAGACGGCGm ψCAAGAAACmψGAACCmψGmψACCmψGAmψCAmψCAAmψmψACmψmψCAAAGGCGGCAAGCmψGCGGmψmψCAAGAAGAmψCAAACCCGAGGCCmψmψC GAGGCmψAACAGAmψmψCmψACACCGmψGAmψCAACAAAAAGmψCCGGCGAGAmψCGmψGCCCAmψGGAAGmψGAACmψmψCAACmψmψCGACGACCCCA ACCmψGAmψmψAmψCCmψGCCmψCmψGGCCmψmψCGGCAAGAGACAGGGCAGAGAGmψmψCAmψCmψGGAACGAmψCmψGCmψGAGCCmψGGAAACCGG CmψCmψCmψGAAGCmψGGCCAAmψGGCAGAGmψGAmψCGAGAAAACCCmψGmψACAACAGGAGAACCAGACAGGACGAGCCmψGCmψCmψGmψmψmψGmψ GGCCCmψGACCmψmψCGAGAGAAGAGAGGmψGCmψGGACAGCAGCAACAmψCAAGCCCAmψGAACCmψGAmψCGGCGmψGGCCCGGGGCGAGAAmψAmψ CCCmψGCmψGmψGAmψCGCCCmψGACAGACCCmψGAAGGAmψGCCCACmψGAGCAGAmψmψCAAGGACmψCCCmψGGGCAACCCmψACACACAmψCCmψG AGAAmψCGGCGAGAGCmψACAAAGAGAAGCAGAGGACAAmψCCAGGCCAAGAAAGAGGmψGGAACAGAGAAGAGCCGGCGGAmψACmψCmψAGGAAGmψACGCCAGCAAGGCCAAGAAmψCmψGGCCGACGACAmψGGmψCCGAAACACCGCCAGAGAmψCmψGCmψGmψACmψACGCCGmψGACACAGGACGCCAmψ GCmψGAmψCmψmψCGCGAAmψCmψGAGCAGAGGCmψmψCGGCCGGCAGGGCAAGAGAACCmψmψmψAmψGGCCGAGAGGCAGmψACACCAGAAmψGGAA GAmψmψGGCmψCACAGCmψAAACmψGGCCmψACGAGGGACmψGAGCAAGACCmψACCmψGmψCCAAAACACmψGGCCCAGmψAmψACCmψCCAAGACCmψ GCAGCAAmψmψGCGGCmψmψCACCAmψCACCAGCGCCGACmψACGACAGAGmψGCmψGGAAAAGCmψCAAGAAAACCGCCACCGGCmψGGAmψGACCAC CAmψCAACGGCAAAGAGCmψGAAGGmψmψGAGGGCCAGAmψCACCmψACmψACAACAGGmψACAAGAGGCAGAACGmψCGmψGAAGGAmψCmψGAGCGm ψGGAACmψGGACAGACmψGAGCGAAGAGAGCGmψGAACAACGACAmψCAGCAGCmψGGACAAAGGGCAGAmψCAGGCGAGGCmψCmψGAGCCmψGCmψG AAGAAGAGGmψmψmψAGCCACAGACCmψGmψGCAAGAGAAGmψmψCGmψGmψGCCmψGAACmψGCGGCmψmψCGAGACACACGCCGCmψGAACAGGCmψG CCCmψGAACAmψmψGCCAGAAGCmψGGCmψGmψmψCCmψGAGAAGCCAAGAGmψACAAGAAGmψACCAGACCAACAAGACCACCGGCAACACCGACAAG AGGGCCmψmψmψGmψGGAAACCmψGGCAGAGCmψmψCmψACAGAAAAAAGCmψGAAAGAAGmψCmψGGAAGCCCGCCGmψGCGAmψCGGGCGGmψmψCC GGCGGAGGmψmψCCACmψAGmψAmψGAACAAmψmψCCCAGGGAAGAGmψGACCmψmψCGAGGAmψGmψCACmψGmψGAACmψmψCACCCAGGGGGAGmψ GGCAGCGGCmψGAAmψCCCGAACAGAGAAACmψmψGmψACAGGGAmψGmψGAmψGCmψGGAGAAmψmψACAGCAACCmψmψGmψCmψCmψGmψGGGACAA GGGGAAACCACCAAACCCGAmψGmψGAmψCmψmψGAGGmψmψGGAACAAGGAAAGGAGCCAmψGGmψmψGGAGGAAGAGGAAGmψGCmψGGGAAGmψGG CCGmψGCAGAAAAAAAmψGGGGACAmψmψGGAGGGCAGAmψmψmψGGAAGCCAAAGGAmψGmψGAAAGAGAGmψCmψCACmψAGmψCCAAAAAAGAAGAG AAAGGmψAGAmψmψACAAAGAmψGACGAmψGACAAAGACmψACAAGGAmψGAmψGAmψGAmψAAGGGAmψCCGGCmψGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3099 LTRP5-ZIM3-KRAB GACCGGCCGCCACCAmψGGCCCCAAAGAAGAAGCGGAAGGmψCmψCmψAGAAmψGAACCAmψGACCAGGAAmψmψmψGACCCCCCAAAGGmψmψmψAC CCACCmψGmψGCCAGCmψGAGAAGAGGAAGCCCAmψCCGCGmψGCmψGmψCmψCmψCmψmψmψGAmψGGGAmψmψGCmψACAGGGCmψCCmψGGmψGCm ψGAAGGACCmψGGGCAmψCCAAGmψGGACCGCmψACAmψCGCCmψCCGAGGmψGmψGmψGAGGACmψCCAmψCACGGmψGGGCAmψGGmψGCGGCACC AGGGAAAGAmψCAmψGmψACGmψCGGGGACGmψCCGCAGCGmψCACACAGAAGCAmψAmψCCAGGAGmψGGGGCCCAmψmψCGACCmψGGmψGAmψmψG GAGGCAGmψCCCmψGCAACGACCmψCmψCCAmψmψGmψCAACCCmψGCCCGCAAGGGACmψmψmψAmψGAGGGmψACmψGGCCGCCmψCmψmψCmψmψ mψGAGmψmψCmψACCGCCmψCCmψGCAmψGAmψGCGCGGCCCAAGGAGGGAGAmψGAmψCGGCCCmψmψCmψmψCmψGGCmψCmψmψmψGAGAAmψGmψ GGmψGGCCAmψGGGCGmψmψAGmψGACAAGAGGGACAmψCmψCGCGAmψmψmψCmψmψGAGmψCmψAACCCCGmψGAmψGAmψmψGACGCCAAAGAAG mψGmψCmψGCmψGCACACAGGGCCCGmψmψACmψmψCmψGGGGmψAACCmψmψCCmψGGCAmψGAACAGGCCmψmψmψGGCAmψCCACmψGmψGAAmψG AmψAAGCmψGGAGCmψGCAAGAGmψGmψCmψGGAGCACGGCAGAAmψAGCCAAGmψmψCAGCAAAGmψGAGGACCAmψmψACCACCAGGmψCAAACmψ CmψAmψAAAGCAGGGCAAAGACCAGCAmψmψmψCCCCGmψCmψmψCAmψGAACGAGAAGGAGGACAmψCCmψGmψGGmψGCACmψGAAAmψGGAAAGGG mψGmψmψmψGGCmψmψCCCCGmψCCACmψACACAGACGmψGmψCCAACAmψGAGCCGCmψmψGGCGAGGCAGAGACmψGCmψGGGCCGGmψCGmψGGA GCGmψGCCGGmψCAmψCCGCCACCmψCmψmψCGCmψCCGCmψGAAGGAAmψAmψmψmψmψGCmψmψGmψGmψGmψCmψAGCGGCAAmψAGmψAACGCmψ AACAGCCGCGGGCCGAGCmψmψCAGCAGCGGCCmψGGmψGCCGmψmψAAGCmψmψGCGCGGCAGCCAmψAmψGGGCCCmψAmψGGAGAmψAmψACAAG ACAGmψGmψCmψGCAmψGGAAGAGACAGCCAGmψGCGGGmψACmψGAGCCmψCmψmψCAGAAACAmψCGACAAGGmψACmψAAAGAGmψmψmψGGGCmψ mψCmψmψGGAAAGCGGmψmψCmψGGmψmψCmψGGGGGAGGAACGCmψGAAGmψACGmψGGAAGAmψGmψCACAAAmψGmψCGmψGAGGAGGGACGmψGG AGAAAmψGGGGCCCCmψmψmψGACCmψGGmψGmψACGGCmψCGACGCAGCCCCmψAGGCAGCmψCmψmψGmψGAmψCGCmψGmψCCCGGCmψGGmψACA mψGmψmψCCAGmψmψCCACCGGAmψCCmψGCAGmψAmψGCGCmψGCCmψCGCCAGGAGAGmψCAGCGGCCCmψmψCmψmψCmψGGAmψAmψmψCAmψG GACAAmψCmψGCmψGCmψGACmψGAGGAmψGACCAAGAGACAACmψACCCGCmψmψCCmψmψCAGACAGAGGCmψGmψGACCCmψCCAGGAmψGmψCCG mψGGCAGAGACmψACCAGAAmψGCmψAmψGCGGGmψGmψGGAGCAACAmψmψCCAGGGCmψGAAGAGCAAGCAmψGCGCCCCmψGACCCCAAAGGAAG AAGAGmψAmψCmψGCAAGCCCAAGmψCAGAAGCAGGAGCAAGCmψGGACGCCCCGAAAGmψmψGACCmψCCmψGGmψGAAGAACmψGCCmψmψCmψCCC GCmψGAGAGAGmψACmψmψCAAGmψAmψmψmψmψmψCmψCAAAACmψCACmψmψCCmψCmψmψGGCGGmψmψCCGGCGGAGGAAmψGAACAAmψmψCC CAGGGAAGAGmψGACCmψmψCGAGGAmψGmψCACmψGmψGAACmψmψCACCCAGGGGGAGmψGGCAGCGGCmψGAAmψCCCGAACAGAGAAACmψmψGm ψACAGGGAmψGmψGAmψGCmψGGAGAAmψmψACAGCAACCmψmψGmψCmψCmψGmψGGGACAAGGGGAAACCACCAAACCCGAmψGmψGAmψCmψmψG AGGmψmψGGAACAAGGAAAGGAGCCAmψGGmψmψGGAGGAAGAGGAAGmψGCmψGGGAAGmψGGCCGmψGCAGAAAAAAAmψGGGGACAmψmψGGAGGG CAGAmψmψmψGGAAGCCAAAGGAmψGmψGAAAGAGAGmψCmψCGGAGGGCCGAGCmψCmψGGCGCACCCCCACCAAGmψGGAGGGmψCmψCCmψGCCG GGmψCCCCAACAmψCmψACmψGAAGAAGGCACCAGCGAAmψCCGCAACGCCCGAGmψCAGGCCCmψGGmψACCmψCCACAGAACCAmψCmψGAAGGmψA GmψGCGCCmψGGmψmψCCCCAGCmψGGAAGCCCmψACmψmψCCACCGAAGAAGGCACGmψCAACCGAACCAAGmψGAAGGAmψCmψGCCCCmψGGGACCAGCACmψGAACCAmψCmψGAGCAAGAGAmψCAAGAGAAmψCAACAAGAmψCAGAAGGAGACmψGGmψCAAGGACAGCAACACAAAGAAGGCCGGCAAG ACAGGCCCAmψGAAAACCCmψGCmψCGmψCAGAGmψGAmψGACCCmψGACCmψGAGAGAGCGGCmψGGAAAACCmψGAGAAAGAAGCCCGAGAACAmψCCCmψCAGCCmψAmψCAGCAACACCAGCAGGGCCAACCmψGAACAAGCmψGCmψGACCGACmψACACCGAGAmψGAAGAAAGCCAmψCCmψGCACGm ψGmψACmψGGGAAGAGmψmψCCAGAAAGACCCCGmψGGGCCmψGAmψGAGCAGAGmψmψGCmψCAGCCmψGCCAGCAAGAAGAmψCGACCAGAACAAGC mψGAAGCCCGAGAmψGGACGAGAAGGGCAAmψCmψGACCACAGCCGGCmψmψmψGCCmψGCmψCmψCAGmψGmψGGCCAGCCmψCmψGmψmψCGmψGmψ ACAAGCmψGGAACAGGmψGmψCCGAGAAAGGCAAGGCCmψACACCAACmψACmψmψCGGCAGAmψGmψAACGmψGGCCGAGCACGAGAAGCmψGAmψm ψCmψGCmψGGCCCAGCmψGAAACCmψGAGAAGGACmψCmψGAmψGAGGCCGmψGACCmψACAGCCmψGGGCAAGmψmψmψGGACAGAGAGCCCmψGGAC mψmψCmψACAGCAmψCCACGmψGACCAAAGAAAGCACACACCCGmψGAAGCCCCmψGGCmψCAGAmψCGCCGGCAAmψAGAmψACGCCmψCmψGGAC CmψGmψGGGCAAAGCCCmψGmψCCGAmψGCCmψGCAmψGGGAACAAmψCGCCAGCmψmψCCmψGAGCAAGmψACCAGGACAmψCAmψCAmψCGAGCACC AGAAGGmψGGmψCAAGGGCAACCAGAAGAGACmψGGAAAGCCmψGAGGGAGCmψGGCCGGCAAAGAGAACCmψGGAAmψACCCCAGCGmψGACCCmψG CCmψCCmψCAGCCmψCACACAAAAGAAGGCGmψGGACGCCmψACAACGAAGmψGAmψCGCCAGAGmψGAGAAmψGmψGGGmψCAACCmψGAACCmψGmψ GGCAGAAGCmψGAAACmψGmψCCAGGGACGACGCCAAGCCmψCmψGCmψGAGACmψGAAGGGCmψmψCCCmψAGCmψmψCCCmψCmψGGmψGGAAAGA CAGGCCAAmψGAAGmψGGAmψmψGGmψGGGACAmψGGmψCmψGCAACGmψGAAGAAGCmψGAmψCAACGAGAAGAAAGAGGAmψGGCAAGGmψmψmψmψ CmψGGCAGAACCmψGGCCGGCmψACAAGAGACAAGAAGCCCmψGAGGCCmψmψACCmψGAGCAGCGAAGAGGACCGGAAGAAGGGCAAGAAGmψmψCGCCAGAmψACCAGCmψGGGCGACCmψGCmψGCmψGCACCmψGGAAAAGAAGCACGGCGAGGACmψGGGGCAAAGmψGmψACGAmψGAGGCCmψGGGAGAG AAmψCGACAAGAAGGmψGGAAGGCCmψGAGCAAGCACAmψmψAAGCmψGGAAGAGGAAAGAAGGAGCGAGGACGCCCAAmψCmψAAAGCCGCmψCmψG ACCGamψmψGGCmψGAGAGCCAAGGCCAGCmψmψmψGmψGAmψCGAGGGCCmψGAAAGAGGCCGACAAGGACGAGmψmψCmψGCAGAmψGCGAGCmψGA AGCmψGCAGAAGmψGGmψACGGCGAmψCmψGAGAGGCAAGCCCmψmψCGCCAmψmψGAGGCCGAGAACAGCAmψCCmψGGACAmψCAGCGGCmψmψCA GCAAGCAGmψACAACmψGCGCCmψmψCAmψmψmψGGCAGAAAGACGGCGmψCAAGAAACmψGAACCmψGmψACCmψGAmψCAmψCAAmψmψACmψmψCA AAGGCGGCAAGCmψGCGGmψmψCAAGAAGAmψCAAACCCGAGGCCmψmψCGAGGCmψAACAGAmψmψCmψACACCGmψGAmψCAACAAAAAGmψCCGGC GAGAmψCGmψGCCCAmψGGAAGmψGAACmψmψCAACmψmψCGACGACCCCAACCmψGAmψmψAmψCCmψGCCmψCmψGGCCmψmψCGGCAAGAGACAGG GCAGAGAGmψmψCAmψCmψGGAACGAmψCmψGCmψGAGCCmψGGAAACCGGCmψCmψCmψGAAGCmψGGCCAAmψGGCAGAGmψGAmψCGAGAAAACC CmψGmψACAACAGGAGAACCAGACAGGACGAGCCmψGCmψCmψGmψmψmψGmψGGCCCmψGACCmψmψCGAGAGAAGAGAGGmψGCmψGGACAGCAGCA ACAmψCAAGCCCAmψGAACCmψGAmψCGGCGmψGGCCCGGGGCGAGAAmψAmψCCCmψGCmψGmψGAmψCGCCCmψGACAGACCCmψGAAGGAmψGCCCACmψGAGCAGAmψmψCAAGGACmψCCCmψGGGCAACCCmψACACACAmψCCmψGAGAAmψCGGCGAGAGCmψACAAAAGAGAAGCAGAGGACAAmψCCA GGCCAAGAAAGAGGmψGGAACAGAGAAGAGCCGGCGGAmψACmψCmψAGGAAGmψACGCCAGCAAGGCCAAGAAmψCmψGGCCGACGACAmψGGmψCC GAAACACCGCCAGAGAmψCmψGCmψGmψACmψACGCCGmψGACACAGGACGCCAmψGCmψGAmψCmψmψCGCGAAmψCmψGAGCAGAGGCmψmψCGGCC GGCAGGGCAAGAGAACCmψmψmψAmψGGCCGAGAGGCAGmψACACCAGAAmψGGAAGAmψmψGGCmψCACAGCmψAAACmψGGCCmψACGAGGGACmψ GAGCAAGACCmψACCmψGmψCCAAAACACmψGGCCCAGmψAmψACCmψCCAAGACCmψGCAGCAAmψmψGCGGCmψmψCACCAmψCACCAGCGCCGACm ψACGACAGAGmψGCmψGGAAAAGCmψCAAGAAAACCGCCACCGGCmψGGAmψGACCACCAmψCAACGGCAAAGAGCmψGAAGGmψmψGAGGGCCAGAm ψCACCmψACmψACAACAGGmψACAAGAGGCAGAACGmψCGmψGAAGGAmψCmψGAGCGmψGGAACmψGGACAGACmψGAGCGAAGAGAGCGmψGAACAA CGACAmψCAGCAGCmψGGACAAAGGGCAGAmψCAGGCGAGGCmψCmψGAGCCmψGCmψGAAGAAGAGGmψmψmψAGCCACAGACCmψGmψGCAAGAGA AGmψmψCGmψGmψGCCmψGAACmψGCGGCmψmψCGAGACACACGCCGCmψGAACAGGCmψGCCCmψGAACAmψmψGCCAGAAGCmψGGCmψGmψmψCCm ψGAGAAGCCAAGAGmψACAAGAAGmψACCAGACCAACAAGACCACCGGCAACACCGACAAGAGGGCCmψmψmψGmψGGAAACCmψGGCAGAGCmψmψC mψACAGAAAAAAGCmψGAAAGAAGmψCmψGGAAGCCCGCCGmψGCGAmψCGGGCGGmψmψCCGGCGGAGGmψmψCCACmψAGmψCCAAAAAAGAAGAGA AAGGmψAGAmψmψACAAAGAmψGACGAmψGACAAAGACmψACAAGGAmψGAmψGAmψGAmψAAGGGAmψCCGGCmψGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3100 Example 6 : CpG- depletion of DNA encoding the guide RNA scaffold improves CasX- mediated editing in vitro

Pathogen-associated molecular patterns (PAMPs), such as unmethylated CpG motifs, are small molecule motifs that are conserved within microbial classes. They are recognized by toll-like receptors (TLRs) and other pattern recognition receptors in eukaryotic cells and often induce nonspecific immune activation. In the case of gene therapy, therapeutic agents containing PAMPs are often not well tolerated and are rapidly cleared from patients under the circumstances of the potent immune response triggered, which ultimately leads to reduced efficacy of the treatment. CpG motifs are short single-stranded DNA sequences containing the dinucleotide CG. When these CpG motifs are unmethylated, they act as PAMPs and thus stimulate immune responses. In this example, experiments were performed to deplete CpG motifs in the guide scaffold coding sequence in the context of an AAV construct encoding CasX variant 491, guide scaffold variant 235, and spacer 7.37 targeting the endogenous B2M (beta-2-microglobulin) locus, and the effect of CpG depletion in the guide scaffold in vitro on editing of the B2M locus was tested. Materials and Methods: Design of CpG-depleted guide scaffold:

Nucleotide substitutions were rationally designed to replace natural CpG motifs within the basic gRNA scaffold variant (gRNA scaffold 235), with the intention of maintaining editing activity while reducing scaffold immunogenicity. It is believed that as many CpG-motifs as possible should be removed from the scaffold coding sequence in order to sufficiently reduce immunogenicity. Scaffold 235 contains a total of eight CpG elements; six of which are predicted to perform base pairing and form complementary strands of the double-stranded secondary structure (see Figure 22). Therefore, the six base-paired CpGs that form three pairs were mutated in concert to maintain important secondary structure. This reduced the number of independent CpG-containing regions to five (three pairs and two single CpGs) to be considered independently for CpG removal. Specifically, mutations were designed in (1) the pseudostem, (2) the scaffold stem, (3) the extended stem vesicle, (4) the extension step, and (5) the extended stem loop, as illustrated in FIG. 23 and described in detail below.

In the pseudostem (region 1), the CpG pairs were flipped to GpC to minimize changes in base composition and sequence. Based on previous experiments involving substitutions of individual base pairs, it was expected that this mutation would not compromise the structure and function of the guide RNA scaffold.

Similarly, in the scaffold stem (region 2), the CpG pair was flipped to GpC to minimize the variation in the base composition and sequence. It was expected that this mutation might impair the structure and function of the guide RNA scaffold, as strong sequence conservation was found in this region in previous experiments that mutated individual bases or base pairs. This strong sequence conservation may be due to the important role of the scaffold stem loop in interacting with the CasX protein and forming a triple helical structural element with the pseudoknot region.

In the extension stem vesicle (Region 3), a single CpG is removed by one of three strategies. First, the vesicle is deleted by a CG->C mutation. Second, the vesicle is regressed by a CG->CT mutation to restore ideal base pairing. Third, the entire extension stem loop is replaced with the extension stem loop of scaffold 174. It should be noted that the replacement of the extension stem loop with the extension stem loop of scaffold 174 itself reproduces scaffold 316 (which has been previously shown to be able to perform efficient editing). There is no CpG motif in the extension stem loop of scaffold 174. Therefore, replacement of the extension stem loop with the extension stem loop of scaffold 174 also removes the CpG motif in the extension stem (Region 4). Based on previous experiments showing that the elongation stem is relatively robust to small changes, we expected that mutations in the elongation stem vesicle might impair the structure and function of the guide RNA scaffold.

In the extension stem (region 4), the CpG pair cannot be flipped to GpC without generating an additional CpG motif. Therefore, the CpG becomes GG and a complementary CC motif. Similar to region 3, the extension stem is relatively robust to small changes, so it is expected that this mutation is unlikely to damage the structure and function of the guide RNA scaffold.

Finally, the extended stem loop (region 5) is mutated in one of three ways based on a previous experimental design to examine the stability of the stem loop. In detail, several variations of the stem loop have been shown to have similar levels of stability, and some of these variations of the stem loop do not contain CpG. Based on these findings, first, the loop is replaced with a new loop with a CUUG sequence. Secondly, the loop is replaced with a new loop with a GAAA sequence. Since the GAAA loop replacement will produce a new CpG adjacent to the loop, it is combined with a C->G base pair exchange on the complementary strand and a corresponding G🡪C base pair exchange, ultimately forming a CUUCGG->GGAAAC exchange. Furthermore, the loop was mutated by inserting an A to interrupt the CpG motif and thereby increase the loop size from 4 to 5 bases. It is expected that random mutations in the extended stem loop will likely have a negative impact on secondary structural stability and therefore on editing. However, the risk associated with substitutions is considered low due to reliance on previously confirmed sequences.

To generate a guide RNA scaffold with reduced CpG levels encoded by DNA, the mutations described above were combined in various configurations. Table 33 below summarizes the combination of mutations used. In Table 33, 0 indicates that no mutation is introduced into the specified region, 1, 2 or 3 indicate that a mutation is introduced in the region, as illustrated in Figure 23, and n/a indicates that it is not applicable. Specifically, for region 1 pseudostem, 1 indicates that a CG->GC mutation is introduced. For region 2 scaffold stems, 1 indicates that a CG->GC mutation is introduced. For region 3 extension stem vesicles, 1 indicates that the vesicle is removed by deleting the bases G and A that form the vesicle, 2 indicates that the vesicle is eliminated by CG->CU mutation to allow base pairing between bases A and U, and 3 indicates that the extension stem loop is replaced with the extension step loop from the guide scaffold 174. For the region 4 extended stem, 1 indicates the introduction of a CG->GC mutation. For the region 5 extended stem loop, 1 indicates the loop was replaced by UUCG->CUUG, 2 indicates the loop was replaced with a base pair adjacent to the loop from CUUCGG->GGAAAC, and 3 indicates the insertion of an A between C and G. Table 33 : Summary of mutations directing CpG reduction and depletion in scaffold 235 Bracket ID Zone 1 ( pseudostem) Zone 2 ( Stent Stem) Zone 3 ( Extended vesicles) Zone 4 ( Extended stems ) Zone 5 ( Extended stem ring) 320 1 0 0 1 0 321 1 0 1 1 0 322 1 0 2 1 0 323 1 0 3 n/a 0 324 1 0 1 1 1 325 1 0 2 1 1 326 1 0 3 n/a 1 327 1 0 1 1 2 328 1 0 2 1 2 329 1 0 3 n/a 2 330 1 0 1 1 3 331 1 0 2 1 3 332 1 0 3 n/a 3 334 1 1 2 1 1 335 1 1 3 n/a 1 336 1 1 1 1 2 337 1 1 2 1 2 338 1 1 3 n/a 2 339 1 1 1 1 3 340 1 1 2 1 3 341 1 1 3 n/a 3 235 0 0 0 0 0

Table 34 below lists the DNA sequences of the designed CpG-reduced or -depleted guide scaffolds. Table 34 : DNA sequences encoding CpG- reduced or -depleted guide RNA scaffolds Bracket ID DNA Sequence SEQ ID NO 320 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCCGCTTAGGGACTTCGGTCCCTAAGAGGCATCAGAG 3210 321 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCCCTTAGGGACTTCGGTCCCTAAGGGCATCAGAG 3211 322 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCCTCTTAGGGACTTCGGTCCCTAAGAGGCATCAGAG 3212 323 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCTCCCTCTTCGGAGGGAGCATCAGAG 3213 324 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCCCTTAGGGACCTTGGTCCCTAAGGGCATCAGAG 3214 325 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCCTCTTAGGGACCTTGGTCCCTAAGAGGCATCAGAG 3215 326 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCTCCCTCCTTGGAGGGAGCATCAGAG 3216 327 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCCCTTAGGGAGGAAACTCCCTAAGGGCATCAGAG 3217 328 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCCTCTTAGGGAGGAAACTCCCTAAGAGGCATCAGAG 3218 329 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGTCCCCTGGAAACAGGGAGCATCAGAG 3219 330 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCCCTTAGGGACTTCAGGTCCCTAAGGGCATCAGAG 3220 331 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCCTCTTAGGGACTTCAGGTCCCTAAGAGGCATCAGAG 3221 332 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGCGACTATGTCGTAGTGGGTAAAGCTCCCTCTTCAGGAGGGAGCATCAGAG 3222 333 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGGCACTATGTGCTAGTGGGTAAAGCCCTTAGGGACCTTGGTCCCTAAGGGCATCAGAG 3223 334 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGGCACTATGTGCTAGTGGGTAAAGCCTCTTAGGGACCTTGGTCCCTAAGAGGCATCAGAG 3224 335 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGGCACTATGTGCTAGTGGGTAAAGCTCCCTCCTTGGAGGGAGCATCAGAG 3225 336 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGGCACTATGTGCTAGTGGGTAAAGCCCTTAGGGAGGAAACTCCCTAAGGGCATCAGAG 3226 337 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGGCACTATGTGCTAGTGGGTAAAGCCTCTTAGGGAGGAAACTCCCTAAGAGGCATCAGAG 3227 338 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGGCACTATGTGCTAGTGGGTAAAGTCCCCTGGAAACAGGGAGCATCAGAG 3228 339 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGGCACTATGTGCTAGTGGGTAAAGCCCTTAGGGACTTCAGGTCCCTAAGGGCATCAGAG 3229 340 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGGCACTATGTGCTAGTGGGTAAAGCCTCTTAGGGACTTCAGGTCCCTAAGAGGCATCAGAG 3230 341 ACTGGGCCTTCTATCTGATTACTCTGAGGCCCATCACCAGGCACTATGTGCTAGTGGGTAAAGCTCCCTCTTCAGGAGGGAGCATCAGAG 3231 Generation of CpG-depleted AAV plasmids:

In addition to AAV2 ITRs, CpG-reduced or -depleted gRNA scaffolds were tested in the context of additional CpG-depleted AAV vectors. Specifically, nucleotide substitutions to replace native CpG motifs in AAV components were designed in silico based on homologous nucleotide sequences from relevant species of the following elements: mouse U1a snRNA (small nuclear RNA) gene promoter, bGHpA (bovine growth hormone polyadenylation) sequence, and human U6 promoter. The coding sequence of CasX 491 was codon-optimized for CpG depletion. All resulting sequences (Tables 34 and 35) were ordered as gene fragments with overhangs suitable for cloning and isothermal assembly to replace the corresponding elements of the existing base AAV plasmid (construct ID 183) individually. The spacer 7.37 (GGCCGAGAUGUCUCGCUCCG; SEQ ID NO: 3137) targeting the endogenous B2M gene was used in the experiments described in this example. When the experiment was first performed ("N=1"), a sample with the non-targeted spacer 0.0 was also included as a control (CGAGACGUAAUUACGUCUCG, SEQ ID NO: 3232; see FIG. 24).

The resulting AAV constructs were generated using standard molecular cloning techniques. The cloned and sequence-verified plasmid constructs were pre-treated for subsequent nucleofection and AAV vector production. In addition to the sequence encoding the gRNA (Table 33), the sequences of the additional components of the AAV constructs are listed in Table 35. Table 35 : Sequences of AAV elements ( 5'-3' in AAV constructs ) element DNA Sequence SEQ ID NO: AAV2 5' ITR CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 3233 CpG-depleted U1a promoter AATGGAGGTGGTACTATGTAGATGAGAATTCAGGAGCAAACTGGGAAAAGCAACTGCTTCCAAATATTTGTGATTTTTACAGTGTAGTTTTGGAAAAACTCTTAGCCTACCAATTCTTCTAAGTGTTTTAAAATGTGGGAGCCAGTACACATGAAGTTATAGAGTGTTTTAATGAGGCTTAAATATTTACTGTAACTATGAAATGCTACACATATCATGCTGTTCAGGCTCTGTGGCCATGCAACTCATACT 3234 CpG depletion of cMycNLS-CasX491-cMycNLS ATGGCTCCAGCTGCCAAGAGAGTGAAGCTGGACTCTAGACAAGAGATCAAGAGGATCAACAAGATTAGAAGAAGGCTGGTCAAGGACAGCAACACCAAGAAGGCAGGCAAGACAGGCCCCATGAAGACCCTGCTTGTCAGAGTGATGACCCCTGACCTGAGAGAGAGACTGGAAAACCTGAGAAAGA AGCCTGAGAACATCCCTCAGCCTATCTCCAACACCAGCAGAGCCAACCTGAACAAGCTGCTGACAGACTACACAGATGAAGAAAGCCATCCTGCATGTGTACTGGGAAGAGTTCCAGAAAGACCCTGTGGGCCTGATGAGCAGAGTGGCCCAGCCTGCCAGCAAGAAGATTGACCAGAACAAACTG AAGCCAGAGATGGATGAGAAGGGCAACCTGACCACAGCTGGCTTTGCCTGCTCTCAGTGTGGCCAGCCTCTGTTTGTGTACAAGCTGGAACAAGTGTCTGAGAAAGGCAAGGCCTACACCAACTACTTTGGCAGATGCAATGTGGCTGAGCATGAGAAGCTGATCCTGCTGGCCCAGCTGAAGCCTGA AAAGGACTCTGATGAGGCTGTGACCTACAGCCTGGGCAAGTTTGGACAGAGAGCCCTGGACTTCTACAGCATCCATGTGACCAAAGAAAGCACACACCCTGTGAAGCCCCTGGCTCAGATTGCTGGCAACAGATATGCCTCTGGACCTGTGGGCAAAGCCCTGTCTGATGCCTGCATGGGAACCATTG CCAGCTTTCTGAGCAAGTACCAGGACATCATCATTGAGCACCAGAAAGTGGTCAAGGGGAACCAGAAGAGGCTGGAATCTCTGAGAGAGCTGGCTGGCAAAGAGAACCTGGAATACCCCTCTGTGACCCTGCCTCCTCAGCCTCACACCAAAGAGGGTGTTGATGCCTACAATGAAGTGATTGCCAGA GTCAGGATGTGGGTCAACCTGAATCTGTGGCAGAAGCTGAAGCTGAGCAGGGATGATGCCAAGCCTCTGCTGAGACTGAAGGGCTTCCCTAGCTTCCCACTGGTGGAAAGGCAGGCTAATGAAGTGGATTGGTGGGACATGGTCTGCAATGTGAAGAAACTGATCAATGAGAAGAAAGAGGATGGCAA GGTTTTCTGGCAGAACCTGGCTGGCTACAAGAGGCAAGAAGCCCTGAGGCCTTACCTGTCCTCTGAGGAAGATAGGAAGAAGGGGAAGAAGTTTGCCAGATACCAGCTGGGAGATCTGCTGCTGCACCTGGAAAAGAAACATGGGGAAGATTGGGGCAAAGTGTATGATGAGGCCTGGGAGAGAATTG ACAAGAAGGTGGAAGGCCTGAGCAAGCACATTAAGCTGGAAGAGGAAAGAAGATCTGAGGATGCCCAGAGCAAGGCTGCCCTGACTGATTGGCTGAGAGCCAAGGCCAGCTTTGTGATTGAGGGCCTGAAAGAGGCTGACAAGGATGAGTTCTGTAGATGTGAACTGAAGCTGCAAAAGTGGTATGGG GACCTGAGGGGCAAGCCCTTTGCCATTGAGGCAGAGAACAGCATCCTGGACATCAGTGGCTTCAGCAAGCAGTACAACTGTGCCTTCATTTGGCAGAAGGATGGGGTTAAGAAGCTCAACCTGTACCTGATCATCAACTACTTCAAAGGTGGCAAGCTGAGGTTCAAGAAGATCAAGCCAGAGGCCT TTGAGGCCAACAGGTTCTACACAGTGATCAACAAAAAGTCTGGGGAGATTGTGCCCATGGAAGTGAACTTCAACTTTGATGATCCCAACCTCATCATCCTGCCACTGGCCTTTTGGCAAGAGACAGGGCAGAGAATTCATCTGGAATGACCTGCTGAGCCTGGAAACAGGCAGCCTGAAACTGGCCAAT GGCAGAGTGATTGAGAAAACCCTGTACAACAGAAGGACCAGACAGGATGAGCCTGCACTGTTTGTGGCCCTGACCTTTGAGAGAAGGGAAGTGCTGGATAGCAGCAACATCAAGCCCATGAACCTGATTGGAGTGGACAGAGGGGAGAATATCCCTGCTGTCATTGCCCTGACAGACCCTGAGGGCTG TCCCCTGAGCAGATTCAAGGACTCCCTGGGCAACCCCACACACATTCTGAGAATTGGGGAGAGCTACAAAGAGAAGCAGAGAACCATCCAGGCCAAGAAAGAAGTGGAACAGAGGAGAGCTGGAGGCTACTCTAGGAAGTATGCCAGCAAAGCCAAGAATCTGGCTGATGACATGGTCAGAAACACAG CCAGGGACCTGCTGTACTATGCTGTGACCCAGGATGCCATGCTGATCTTTGAGAATCTGAGCAGAGGATTTGGGAGACAAGGCAAGAGGACCTTCATGGCTGAAAGACAGTACACCAGGATGGAAGATTGGCTGACTGCCAAACTGGCCTATGAGGGACTGAGCAAGACCTACCTGTCCAAGACACTG GCCCAGTATACCTCCAAGACCTGCAGCAATTGTGGCTTCACCATCACCTCTGCAGACTATGACAGAGTGCTGGAAAAGCTCAAGAAAACAGCCACAGGCTGGATGACCACCATCAATGGGAAAGAGCTGAAGGTTGAGGGCCAGATCACCTACTACAACAGATACAAAAGGCAGAATGTGGTTAAGGA CCTGTCTGTGGAACTGGACAGACTGTCTGAGGAATCTGTGAACAATGACATCAGCAGCTGGACCAAGGGCAGATCTGGGGAAGCTCTGAGCCTGCTGAAGAAGAGATTCAGCCACAGACCTGTGCAAGAGAAGTTTGTGTGTCTGAACTGTGGCTTTGAGACACATGCTGATGAACAGGCTGCTCTGA ACATTGCCAGAAGCTGGCTGTTCCTGAGAAGCCAAGAGTACAAGAAGTACCAGACCAACAAGACCACAGGCAACACTGACAAGAGGGCCTTTGTGGAAACCCTGGCAGAGCTTCTACAGAAAGAAACTCAAAGAAGTCTGGAAGCCTGCTGTGGGCAGCCCTGCAGCTAAAAGGGTCAAGCTGGACTGA 3235 CpG-depleted bGH-polyadenylation sequence CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCAGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTCCTAATAAAATGAGGAAATTGCATCACATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGA 3236 CpG-depleted U6 promoter GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACAATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATATTTGATGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCATAACTTGAAAGTATTTCTATTTCTTGGCTTTATATATCTTGTGGAAAGGAC 3237 See sgRNA sequences in Table 34 above . AAV2 3' ITR AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 3238 AAV production:

On the day of transfection, HEK293T cells maintained in suspension in FreeStyle 293 medium were seeded at 1.5E6 cells/mL in 20-30 mL of medium. Endotoxin-free pAAV plasmids with the transgene flanked by ITR repeats were co-transfected with plasmids providing adenoviral helper genes for replication and the AAV rep/cap genome using PEI-Max (Polysciences) in serum-free Opti-MEM medium. Three days later, the culture was centrifuged to separate the supernatant from the cell pellet, and the AAV particles were collected, concentrated, and filtered according to standard procedures.

To determine the viral genome (vg) titer, 1 µL of crude lysate virus was digested with DNase and ProtK, followed by quantitative PCR. 5 µL of digested virus was used in a 25 µL qPCR reaction consisting of IDT primetime master mix, a set of primers and 6'FAM/Zen/IBFQ probe (IDT), designed to amplify a 62 bp-fragment located in the AAV2-ITR. AAV ITR plasmid was used as a reference standard to calculate the titer (vg/mL) of the viral samples. In vitro AAV transduction of induced neurons:

24 hours before transduction, induced neurons were seeded at 50,000 per well on a 96-well plate coated with Matrigel. AAV expressing the CasX:gRNA system with various types of guide scaffolds were then diluted in neuron plating medium and added to the cells. For the first experiment ("N=1"), cells were transduced at an infection multiplicity (MOI) of 4e3 viral genomes (vg)/cell (see Figure 24). Seven days after plating, induced neurons were transduced with virus diluted in fresh feed medium. Eight days after transduction, cells were extracted using lysis buffer, 4-well replicates were pooled according to the experimental conditions, and genomic DNA (gDNA) was collected and prepared for analysis of edits at the B2M locus using next-generation sequencing (NGS). For the second run ("N=2"), cells were transduced at an MOI of 3e3 vg/cell, 1e3 vg/cell, or 3e2 vg/cell (see Figures 25, 26, and 27). Seven days after plating, induced neurons were transduced with virus diluted in fresh feed medium. Seven days after transduction, cells were extracted using lysis buffer, 2-well replicates were pooled according to experimental conditions, and gDNA was collected and prepared for analysis of edits at the B2M locus using NGS. Samples without AAV transduction were included as controls. NGS processing and analysis:

Genomic DNA (gDNA) from collected cells was extracted using the Zymo Quick-DNA Miniprep Plus kit according to the manufacturer's instructions. Target amplicon was formed by amplifying the region of interest from 200 ng of extracted gDNA with a set of primers specific for the human B2M locus. These gene-specific primers contain additional sequences at the 5' end to introduce the Illumina adapter and a 16-nucleotide unique molecular identifier. The amplified DNA product was purified using the Ampure XP DNA purification kit. The quality and quantification of the amplicon was assessed using the Fragment Analyzer DNA analysis kit (Agilent, dsDNA 35-1500 bp). The amplicon was sequenced on the Illumina Miseq according to the manufacturer's instructions. The raw fastq files were quality controlled and processed using cutadapt v2.1, flash2 v2.2.00, and CRISPResso2 v2.0.29. Each sequence was quantified for insertions or deletions (indels) relative to the reference sequence within a window around the 3' end of the spacer (a 30 bp window centered at -3 bp from the 3' end of the spacer). For each sample, CasX activity was quantified as the total percentage of reads containing insertions, substitutions, and/or deletions anywhere within this window. Results:

Mutations were introduced into guide scaffold 235 to reduce the CpG content of the DNA sequence encoding the guide scaffold. Surprisingly, both CpG-reduced and CpG-depleted scaffold variants produced higher editing levels in induced neurons compared to scaffold 235. This was the case for two independent experimental repetitions (the results of the first experimental repetition are shown in Figure 24, and the results of the second experimental repetition are shown in Figures 25 to 27) and multiple MOIs (see Figures 26 to 27). The enhanced editing levels are unexpected because the purpose of reducing the CpG content is only to maintain editing activity while reducing immunogenicity. In fact, the mutation enhances editing activity, rather than just maintaining it.

Notably, scaffold 320 showed a significant improvement in potency compared to scaffold 235. Scaffold 320 included mutations to only two regions of the scaffold: the pseudostem and the extension stem (regions 1 and 4). In addition, some combinations of mutations produced edits that were worse than scaffold 320. However, even CpG-reduced scaffolds that performed worse than scaffold 320, such as scaffolds 331 and 334, performed similarly or better than scaffold 235.

Based on these results, without wishing to be bound by theory, it is believed that the potency enhancement seen in many CpG-reduced and CpG-depleted scaffolds may be caused by a mutation present in all CpG-reduced scaffolds (i.e., regions 1 and/or 4). Since mutations to region 4 are absent in scaffolds with extended stem loop substitutions (i.e., the third mutation to region 3), and these scaffolds show potency improvements over 235 similar to 320, it is believed that the beneficial effects may be caused by mutations in region 1 (pseudostem), which are present in all scaffolds tested. Additional experiments will be performed to test the effects of individual mutations in the pseudostem (region 1) and extended stem (region 4), respectively.

Additionally, the N=1 data presented in Figure 24 indicate that all new scaffolds carrying mutations in region 2 (scaffold stem) have slightly lower editing levels than their respective counterparts without the mutation. This suggests that mutating this position in the scaffold stem may have a small adverse effect on editing efficacy. This will be tested in additional experiments.

The results described herein demonstrate that introducing mutations that reduce the CpG content of the DNA encoding the guide RNA scaffold improves gene editing relative to guide scaffold 235. Example 7 : Demonstration that inclusion of an ADD domain from DNMT3A enhances the activity and specificity of LTRP molecules

In addition to its C-terminal methyltransferase domain, DNMT3A also contains two N-terminal domains that regulate its function and recruitment to chromatin: the ADD domain and the PWWP domain. The PWWP domain has been reported to interact with methylated histone tails, including H3K36me3. The ADD domain is known to have two key functions: 1) it regulates the catalytic activity of DNMT3A ectopically by acting as a methyltransferase autoinhibitory domain, and 2) it recognizes unmethylated H3K4 (H3K4me0). The interaction of the ADD domain with the H3K4me0 mark reveals the catalytic sites of DNMT3A, thereby recruiting active DNMT3A to chromatin to re-implement methylation at these sites.

In light of these functions of the ADD domain, experiments were performed to assess whether incorporation of the ADD domain into the LTRP5 construct (previously described in Example 1) would result in improved long-term repression of the target locus and reduced off-target methylation. The effects of incorporation of the PWWP domain and the ADD domain on LTRP activity and specificity were also assessed. Materials and Methods: Generation of LTRP Constructs and Plasmid Cloning:

Standard molecular cloning techniques were used to construct plastid constructs encoding variants of the LTRP5 construct with a ZIM3-KRAB domain (LTRP5.A; see Figure 28 for LTRP5 configuration). The resulting constructs contained sequences encoding one of the following four alternative variants of LTRP5-ZIM3, in which an additional DNMT3A domain was incorporated: 1) LTRP5-ZIM3 + ADD; 2) LTRP5-ZIM3 + ADD + PWWP; 3) LTRP5-ZIM3 + ADD, without the DNMT3A catalytic domain; and 4) LTRP5-ZIM3 + ADD + PWWP, without the DNMT3A catalytic domain. The sequences of key elements within the LTRP5-ZIM3 molecule and its variants are listed in Table 36, with the full-length protein sequences of each LTRP5-ZIM3 and its variants listed in Table 37. Figure 29 is a schematic diagram showing various LTRP5 constructs analyzed in this example. The sequence encoding the LTRP molecule also contains a 2×FLAG tag. The plasmid also has a construct encoding a gRNA scaffold variant 174 with a spacer targeting the endogenous B2M locus or a non-targeting control (spacer sequences listed in Table 38). Table 36 : Sequences of LTRP components ( e.g., additional domains fused to dCasX ) used to generate the LTRP5 variants shown in Figure 29 Components Protein sequence SEQ ID NO ZIM3 KRAB domain MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESL 3240 DNMT3A catalytic domain (CD) NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAM GVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV 126 DNMT3L interaction domain MGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQ RPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL 127 dCasX491 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 4 Connector 1 GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE 123 Connector 2 SSGNSNANSRGPSFSSGLVPLSLRGSH 122 Connector 3A' GGSGGG 124 Connector 3B GGSGGGS 120 Connector 4 GSGSGGG 121 NLS A PKKKRKV 30 NLS B DNMT3A ADD domain ERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLLRRREDWPSRLQMFFAN 125 DNMT3A PWWP domain TKAADDEPEYEDGRGFGIGELVWGKLRGFSWWPGRIVSWWMTGRSRAAEGTRWVMWFGDGKFSVVCVEKLMPLSSFCSAFHQATYNKQPMYRKAIYEVLQVASSRAGKLFPACHDSDESDSGKAVEVQNKQMIEWALGGFQPSGPKGLEPPEEEKNPYKEV 3252 Endogenous sequence between DNMT3A PWWP and ADD domains (endo) YTDMWVEPEAAAYAPPPPAKKPRKSTTEKPKVKEIIDERTR 3253 Table 37 : Protein sequences of LTRP5 variants analyzed in this example LTRP ID Protein sequence SEQ ID NO LTRP5-ZIM3 MAPKKKRKVSRMNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGG SPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQ ECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGP SFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGTTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFH RILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLR EYFKYFSQNSLPLGGSGGGMNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQI WKPKDVKESLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGF ACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMG TIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDW WDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAA LTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVIN KKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTR MEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTK GRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSPKKKRKV 3131 LTRP5-ZIM3+ADD MAPKKKRKVSRMERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDP WNCYMCGHKGTYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQE WGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLE LQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLV PLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRP FFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGSGGGMNNSQ GRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLGGPSSGAPPPSGGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPIS NTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEK DSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDA YNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKH GEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKL NLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREV LDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRG FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVN NDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSPKKKRKV 3132 LTRP5-ZIM3 + ADD + PWWP MAPKKKRKVSRMTKAADDEPEYEDGRGFGIGELVWGKLRGFSWWPGRIVSWWMTGRSRAAEGTRWVMWFGDGKFSVVCVEKLMPLSSFCSAFHQATYNKQPMYRKAIYEVLQVASSRAGKLFPACHDSD ESDSGKAVEVQNKQMIEWALGGFQPSGPKGLEPPEEEKNPYKEVYTDMWVEPEAAAYAPPPPAKKPRKSTTEKPKVKEIIDERTRERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQNCKNCF LECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVL KDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMI DAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYF ACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYA LPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGSGGGMNNSQGRV TFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEE FQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAG NRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWD MVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEF CRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLE TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMV RNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDL SVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSPKKKRKV 3254 LTRP5-ZIM3 + ADD - CD MAPKKKRKVSRMERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLLRRREDWPSRLQMFFANSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLF RNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGSGGGM NNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEQEIKRINKIRRRLVKDSNT KKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDE KGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGN RYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGK VYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANG RVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNC GFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSPKKKRKVDYKDDDDKDYKDDDDK 3255 LTRP5-ZIM3 + ADD + PWWP - CD MAPKKKRKVSRMTKAADDEPEYEDGRGFGIGELVWGKLRGFSWWPGRIVSWWMTGRSRAAEGTRWVMWFGDGKFSVVCVEKLMPLSSFCSAFHQATYNKQPMYRKAIYEV LQVASSRAGKLFPACHDSDESDSGKAVEVQNKQMIEWALGGFQPSGPKGLEPPEEEKNPYKEVYTDMWVEPEAAAYAPPPPAKKPRKSTTEKPKVKEIIDERTRERLVYEV RQKCRNIEDICISCGSLNVTLEHPLFIGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGL LRRREDWPSRLQMFFANSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGP FDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQA QVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGSGGGMNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEP WLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP SEQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQ NKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNR YASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFP SFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEE RRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFE ANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGE NIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAE RQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSW TKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSPKKKRKV 3256 Table 38 : Sequences of spacers used in constructs Spacer ID Target gene sequence SEQ ID NO 0.0 Non-targeted CGAGACGUAAUUACGUCUCG 3232 7.37 B2M GGCCGAGAUGUCUCGCUCCG 3137 7.160 B2M UAAACAUCACGAGACUCUAA 3113 7.165 B2M UCCCUAUGUCCUUGCUGUUU 3114 Transfection of HEK293T cells:

The inoculated HEK293T cells were transiently transfected with 100 ng of LTRP5 variant plasmids, each of which contained a LTRP:gRNA construct encoding LTRP5-ZIM3 or one of its alternative variants (Figure 29; see Table 37 for sequences), wherein the gRNA had a non-targeting spacer 0.0 or a spacer targeting B2M (see Table 38 for spacer sequences). Spacers 7.160 and 7.165 have been shown to repress the B2M locus when used with LTRPs but not with dXRs made from dCasX fused to the ZIM3 KRAB domain (data not shown). Each construct was tested in triplicate. 24 hours after transfection, cells were selected for three days with 1 μg/mL puromycin. Cells were harvested for inhibition analysis on days 5, 12, 21, and 51 after transfection. Briefly, inhibition analysis was performed by analyzing B2M protein expression by HLA immunostaining followed by flow cytometry as described in Example 4. In addition, HEK293T cells transiently transfected with LTRP5 variant plasmids and B2M targeting gRNA or non-targeting gRNA were collected seven days after transfection for gDNA extraction for bisulfite sequencing to assess off-target methylation at the VEGFA locus, which was performed as described in Example 5. Results:

The effect of incorporating the ADD domain into the LTRP5 molecule with or without the PWWP domain on increased long-term repression and decreased off-target methylation of the target B2M locus was assessed. Variant forms of the LTRP5-ZIM3 molecule were assessed with B2M -targeting gRNA (with spacer 7.37 and LTRP-specific spacers 7.160 and 7.165) or non-targeting gRNA, and the results are depicted in the graphs in Figures 30 to 33. Figure 30 shows that the use of spacer 7.37 produced saturated levels of repressive activity when paired with LTRP5-ZIM3, LTRP5-ZIM3 + ADD, and LTRP5-ZIM3 + ADD + PWWP, making it more challenging to assess the activity differences between LTRP5 variants. However, when spacers 7.160 and 7.165 were used, the differences in inhibitory activity between LTRP5 variants were more pronounced (Figures 31 and 32). The data show that the incorporation of the ADD domain caused a significant increase in long-term inhibition when paired with two LTRP-specific spacers compared to the levels of inhibition achieved with other LTRP5-ZIM3 molecules. At the same time, the incorporation of both the ADD domain and the PWWP domain did not cause improved inhibition of the B2M locus, especially compared to the baseline LTRP5-ZIM3 molecule. As expected, the two LTRP5 variants without the DNMT3A catalytic domain exhibited poor long-term inhibition. In addition, the results depicted in Figure 33 indicate that the addition of the ADD domain appears to increase specificity, given that a lower percentage of HLA-negative cells were observed relative to the baseline LTRP5-ZIM3 molecule.

Bisulfite sequencing was used to assess off-target CpG methylation at the VEGFA locus that may be mediated by LTRP5 variants. FIG. 34 depicts the results of bisulfite sequencing, which specifically shows the percentage of CpG methylation around the VEGFA locus. The results confirmed that for all B2M -targeting gRNAs as well as non-targeting gRNAs, incorporation of the ADD domain into the LTRP5-ZIM3 molecule significantly reduced the level of off-target methylation at the VEGFA locus ( FIG. 34 ). Figure 35 is a scatter plot that plots the activity-specificity profile of the LTRP5-ZIM3 variants studied in this example, where activity is measured as the average percentage of HLA-negative cells at day 21 when paired with spacer 7.160, and specificity is represented by the percentage of off-target CpG methylation at the VEGFA locus quantified at day 7 when paired with spacer 7.160. The scatter plot clearly shows that the addition of the ADD domain significantly increases the activity of the LTRP5 molecule relative to the baseline ELX5 molecule without the ADD domain (Figure 35).

The experiments demonstrated that including the DNMT3A ADD domain but not the ADD domain and PWWP domain improved the inhibitory activity and specificity of the LTRP molecule. The enhancement of activity and specificity was observed with a variety of gRNAs, demonstrating the importance of incorporating the ADD domain into the LTRP. Example 8 : Demonstration that including the ADD domain from DNMT3A in the LTRP enhances on -target activity and reduces off-target methylation

Experiments were performed to assess the effects of incorporation of the ADD domain into LTRP molecules with configurations 1, 4, and 5 (Figure 1) on long-term repression and off-target methylation of target loci. Materials and Methods: Generation of LTRP constructs and plastid selection:

Standard molecular cloning techniques were used to construct plastid constructs encoding LTRP molecules with configurations 1, 4 and 5, with ZNF10-KRAB or ZIM3-KRAB domains and DNMT3A ADD domains. The sequences of the resulting LTRP molecules are listed in Table 39, which also shows the abbreviated construct names of specific LTRP molecules (e.g., LTRP1.A, 1.B). Figure 36 is a schematic diagram showing the general structure of LTRP molecules with ADD domains incorporated into LTRP configurations 1, 4 and 5. The sequence encoding the LTRP molecule also contains a 2×FLAG marker. The plastid also has a sequence encoding a gRNA scaffold 174 with a spacer targeting the endogenous B2M locus or a non-targeting control (spacer sequence listed in Table 38). Table 39 : Protein sequences of various LTRP1 , 4 and 5 variants analyzed in this example LTRP # domain Protein sequence SEQ ID NO LTRP1 ZNF10-KRAB, DNMT3A ADD, DNMT3A CD, DNMT3L interaction (LTRP1.D) MAPKKKRKVSRMERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKED PWNCYMCGHKGTYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKH IQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTV NDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPS FSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYAL PRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGG PSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGGSGGGSAQEIKRINKIRRRLVKDSNTKKAGKTGPMK TLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVS EKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQK RLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQE ALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLR GKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETG SLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRK YASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTT INGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTD KRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPTSPKKKRKV 3257 ZIM3-KRAB, DNMT3A ADD, DNMT3A CD, DNMT3L interaction (LTRP1.C) MAPKKKRKVSRMERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPW NCYMCGHKGTYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEW GPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLEL QECLEHGRIAKFSKVRTITTTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVP LSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRPF FWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGPSSGAPPPSG GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGGSGGGSAQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLR ERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCN VAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEY PSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQAANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKK FARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFS KQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYY AVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVV KDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRS GGSGGGSTSMNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLTSPKKKRKV 3258 ZNF10-KRAB, DNMT3A CD, DNMT3L interaction (LTRP1.B) MAPKKKRKVSRVNGSGSGGGMNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQE WGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPL ASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV SSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGTTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSS CDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG GSGGGSAQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA QPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHP VKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKL SRDDAKPLLRLKGFPSFPLVERQAANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWE RIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLY LIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALT FERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYY AVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYY NRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETW QSFYRKKLKEVWKPAVRSGGSGGGSTSMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPTSPKKKRKV 3259 ZIM3-KRAB, DNMT3A CD, DNMT3L interaction (LTRP1.A) See the sequence in Table 14 3066 LTRP4 ZNF10-KRAB, DNMT3A ADD, DNMT3A CD, DNMT3L interaction (LTRP4.D) MAPKKKRKVSRMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPGGSGGGSAMERLVYEVRQKCRNIEDICISCGS LNVTLEHPLFIGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLLRRREDWPSRLQMFFANNHD QEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGR LFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKD QHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVL SLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEA VTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTD YTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLG KFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVR MWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKV YDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLII NYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSS NIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGR QGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNN DISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSPKKKRKV 3260 ZIM3-KRAB, DNMT3A ADD, DNMT3A CD, DNMT3L interaction (LTRP4.C) MAPKKKRKVSRMNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLGGSGGG SAMERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKG TYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGG SPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRI AKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMG PMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLL LTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGPSSGAPPPSGGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPI SNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPE KDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVD AYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKK HGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKK LNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERRE VLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSR GFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESV NNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSPKKKRKV 3261 ZNF10-KRAB, DNMT3A CD, DNMT3L interaction (LTRP4.B) MAPKKKRKVSRMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPGGSGGGSAMNHDQEFDPPKVYPP VPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFF EFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIY KTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIF MDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGPSSG APPPSGSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYK LEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQD IIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKK LINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAK ASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEI VPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMED WLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGR SGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSPKKKRKV 3262 ZIM3-KRAB, DNMT3A CD, DNMT3L interaction (LTRP4.A) MAPKKKRKVSRVNGSGSGGGMNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQE WGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRP LASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFAC VSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGS SCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDA PKVDLLVKNCLLPLREYFKYFSQNSLPLGGSGGGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPG GPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPL FVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSK YQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCN VKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWL RAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSG EIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRME DWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKG RSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSPKKKRKV 3263 LTRP5 ZNF10-KRAB, DNMT3A ADD, DNMT3A CD, DNMT3L interaction (LTRP5.D) MAPKKKRKVSRMNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVK ESLGGSGGGSAMNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGS PCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPS FSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFH RILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLRE YFKYFSQNSLPLGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEQEIKRINKIRRRLVKDS NTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAG FACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACM GTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVD WWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKA ALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVI NKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVI ALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYT RMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTK GRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAVRSGGSGGGSTSPKKKRKV 3264 ZIM3-KRAB, DNMT3A ADD, DNMT3A CD, DNMT3L interaction (LTRP5.C) See the sequence in Table 37 3132 ZNF10-KRAB, DNMT3A CD, DNMT3L interaction (LTRP5.B) MAPKKKRKVSRVNGSGSGGGMNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEW GPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLA STVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSS GNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCD RCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVD LLVKNCLLPLREYFKYFSQNSLPLGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGGSG GGSAQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPAS KKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLA QIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAK PLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKV EGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFK GGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVL DSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAML IFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNV VKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKL KEVWKPAVRSGGSGGGSTSSQGSVTFRDVAIDFSQEEWKWLQPAQRDLYRRVMLENYGHLVSLGLSISKPDVVSLLEQGKEPWLGKRNVKRDLFSASESSTSPKKKRKV 3265 ZIM3-KRAB, DNMT3A CD, DNMT3L interaction (LTRP5.A) See the sequence in Table 37 3131 Transfection of HEK293T cells:

The inoculated HEK293T cells were transiently transfected with 100 ng of LTRP variant plasmids, each of which contained an LTRP:gRNA construct encoding an LTRP molecule (Table 39; Figure 36), wherein the gRNA had a non-targeting spacer 0.0 or a spacer targeting B2M (Table 38). Each construct was tested in triplicate. 24 hours after transfection, cells were selected with 1 μg/mL puromycin for 3 days. Cells were collected for inhibition analysis on days 8, 13, 20, and 27 after transfection. Briefly, inhibition analysis was performed by analyzing B2M protein expression by HLA immunostaining followed by flow cytometry as described in Example 4. In addition, cells were also collected on day 5 post-transfection for gDNA extraction for bisulfite sequencing to assess off-target methylation at the non-target VEGFA locus, which was performed using a method similar to that described in Example 5. Results:

The effects of incorporating the ADD domain into LTRP molecules with configurations 1, 4, or 5, with ZNF10 or ZIM-KRAB on long-term repression and off-target methylation of the B2M locus were evaluated. LTRP molecules were tested with B2M targeting gRNA or non-targeting gRNA, and the results are depicted in the graphs in Figures 37 to 47. The data demonstrated that incorporation of the ADD domain into LTRP molecules significantly increased B2M repression at all time points for all LTRP configurations containing ZIM3-KRAB when spacer 7.160 was used (Figure 37), and similar findings were observed when spacers 7.165 and 7.37 were used (data not shown). Figure 38 shows the B2M inhibition obtained after using LTRP5 containing ZNF10 or ZIM3-KRAB when paired with gRNA with spacer 7.160; the data confirm that the inclusion of the ADD domain generally increases persistent B2M inhibition, with LTRP5-ZIM3 + ADD having higher activity than LTRP5-ZNF10 + ADD. Similar time course findings were observed for LTRP1 and LTRP4 and two other spacers (data not shown). Figure 39 shows the B2M inhibition obtained after using LTRP5 containing ZIM3-KRAB when paired with any of the three B2M targeting gRNAs, and the data confirm that the inclusion of the ADD domain generally causes higher B2M inhibition. Similar time course findings were also observed for LTRP1 and LTRP4 (data not shown).

Figures 40-42 show the resulting B2M inhibition at the 27 day time point for all LTRP configurations and gRNAs tested. The results show that the increase in B2M inhibition using the next best spacers 7.160 and 7.165 was more significant than using spacer 7.37. In addition, the use of LTRP1 and LTRP5, which contain DNMT3A and DNMT3L domains at the N-terminus of the molecule, resulted in the greatest increase in B2M inhibition after the addition of the DNMT3A ADD domain (Figures 40-42). The use of LTRP4, which has DNMT3A/3L domains 3' to the KRAB domain and 5' to dCasX, resulted in less activity enhancement, which can be attributed to the reduced ability of the ADD domain to properly interact with chromatin.

The specificity of the LTRP molecules was determined by analyzing the CpG methylation levels at the VEGFA gene (off-target locus) using bisulfite sequencing, and the data are shown in Figures 43 to 51. The data confirmed that including the DNMT3A ADD domain caused a significant reduction in off-target methylation of the VEGFA locus for all conditions tested (Figures 43 to 45). It is worth noting that the increase in specificity mediated by including the ADD domain was most significant for the LTRP1 and LTRP5 configurations, both of which have the DNMT3A/3L domain at the N-terminal end of the molecule. Interestingly, LTRP molecules containing the ZIM3-KRAB domain caused stronger off-target methylation of the VEGFA locus. In addition, even in the absence of the ADD domain, the specificity generated using the LTRP4 and 5 configurations was higher than that using the LTRP1 configuration. Inclusion of the ADD domain in LTRP5-ZIM3 resulted in minimal off-target methylation compared to LTRP1-ZIM3 and LTRP4-ZIM3 configurations.

Figures 46-51 are a series of scatter plots plotting the activity-specificity profiles of various LTRP molecules, where activity is measured as the average percentage of HLA-negative cells at day 27 and specificity is determined by the percentage of off-target CpG methylation at the VEGFA locus at day 5. The data demonstrate that in all three B2M -targeting spacers tested, inclusion of the ADD domain resulted in increased on-target B2M inhibition and decreased off-target methylation at the VEGFA locus. LTRP molecules with configurations 1 and 5 exhibited the greatest increase in activity and specificity at each spacer tested.

The experimental results discussed in this example support the findings in Example 7, namely that the data demonstrate that including the DNMT3A ADD domain enhances the strength of repression at early time points and the heritability of silencing across cell divisions, and reduces off-target methylation induced by the DNMT3A catalytic domain in LTRP molecules. The data also confirm that there are inherent differences in the specificity of different LTRP configurations, which can be exacerbated by the use of more potent repressor subdomains. This reduction in specificity can be alleviated by including the DNMT3A ADD domain, which can also result in more on-target inhibition overall. The increase in repressive activity is believed to be mediated by the function of the DNMT3A ADD domain to recognize H3K4me0 and subsequent recruitment to chromatin. The increased specificity is believed to be mediated by the function of the DNMT3A ADD domain to induce ectopic inhibition of the catalytic domain of DNMT3A in the absence of H3K4me0 binding. The results also highlight that positioning the ADD domain in the different configurations tested is important to achieve the strongest increase in specificity and activity of LTRP molecules. Example 9 : Demonstration of reversibility of silencing of target loci mediated by LTRP molecules using DNMT1 inhibitors

Experiments were performed to demonstrate that persistent repression of the target locus mediated by LTRP molecules is reversible, such that treatment with a DNMT1 inhibitor will remove the methyl mark to reactivate expression of the target gene. Materials and Methods: Generation of LTRP constructs and lentiviral plasmid selection:

Lentiviral plasmid constructs encoding LTRP molecules were constructed using standard molecular cloning techniques. These constructs included sequences encoding the catalytically inactive CasX protein 491 (dCasX491), the KRAB domain from ZNF10 or ZIM3, and the catalytic and interaction domains from DNMT3A (D3A) and DNMT3L (D3L), respectively. Briefly, the constructs were oligonucleotide ordered and assembled by overlapping extension PCR followed by isothermal assembly. The resulting plasmids contained constructs arranged in different configurations to produce LTRP molecules. The sequences encoding the LTRP molecules also contained a 2×FLAG marker. The plasmids also had sequences encoding the gRNA scaffold variant 174 with a spacer targeting the endogenous B2M locus or a non-targeting control. These constructs were all cloned upstream of the P2A puromycin element on the lentiviral plasmid. The cloned and sequence-verified constructs were pre-processed and quality assessed prior to transfection in HEK293T cells.

LTRP5 containing the ZIM3-KRAB domain and CasX variant 491 was used in this experiment. B2M targeting gRNA with scaffold 174 containing spacer 7.37 (SEQ ID NO: 3137) or non-targeting gRNA containing spacer 0.0 (SEQ ID NO: 3232) was used in this experiment. Transfection of HEK293T cells:

HEK293T cells were transfected with 100 ng of plasmids containing constructs encoding CasX 491 or LTRP5 containing the ZIM3-KRAB domain with B2M- targeting gRNA or non-targeting gRNA and cultured for 58 days. These transfected HEK293T cells were then seeded at approximately 30,000 cells per well in a 96-well plate and treated with the DNMT1 inhibitor 5-aza-2'-deoxycytidine (5-azadC) at concentrations ranging from 0 µM to 20 µM. Six days after treatment with 5-azadC, cells were harvested for B2M silencing analysis on days 5, 12, and 21 after transfection. Briefly, inhibition analysis was performed by analyzing B2M protein expression by HLA immunostaining followed by flow cytometry as described in Example 4. Treatment with each dose of 5-azadC was performed in triplicate for each experimental condition. Results:

The graph in Figure 52 shows the percentage of transfected HEK293T cells treated with 5-azadC expressing B2M protein at a specified concentration. The data show that 5-azadC treatment of cells transfected with plasmids encoding LTRP5-ZIM3 and B2M targeting gRNA causes reactivation of the B2M gene (Figure 52). Specifically, approximately 75% of cells treated with 20 µM 5-azadC exhibit B2M expression, compared to 25% of cells with B2M expression at 0 µM concentration (Figure 52). In addition, 5-azadC treatment of cells transfected with plasmids encoding CasX 491 and B2M targeting gRNA did not exhibit reactivation of the B2M gene. Figure 53 is a graph showing B2M inhibitory activity and gene reactivation after 5-azadC treatment side by side. The data showed that B2M inhibition occurred after transfection with CasX 491 or LTRP5-ZIM3 and B2M targeting gRNA, resulting in approximately 75% inhibition of B2M expression at day 58; however, B2M expression increased after 5-azadC treatment (Figure 53). As expected, 5-azadC treatment of cells transfected with CasX 491 or LTRP5-ZIM3 and non-targeting gRNA did not show inhibition or reactivation (Figures 52 to 53).

The experiment confirmed the reversibility of LTRP-mediated repression of the target locus. By using a DNMT1 inhibitor to remove the methyl mark formed by the LTRP molecule, the silenced target gene was reactivated to induce the expression of the target protein. Example 10 : Exemplary sequences of LTRP fusion proteins  

Table 40 provides the sequences of components used in exemplary full-length LTRP fusion proteins in configuration 1, 4 or 5, with an ADD domain (Figure 1), with a human ZIM3 or ZNF10 KRAB domain or one of the following top nine most potent inhibitory subdomains: domain_7694, domain_10123, domain_15507, domain_17905, domain_20505, domain_26749, domain_27604, domain_29304 and domain_30173. In Table 40, for each LTRP configuration, the components are listed in order from N-terminus to C-terminus. Table 40 : Exemplary protein sequences of LTRP fusion proteins LTRP # Components domain AA sequence SEQ ID NO LTRP1 with ADD domain Start codon + NLS + buffer sequence MAPKKKRKVSR 3269 Start codon + DNMT3A ADD domain MERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLLRRREDWPSRLQMFFAN 3270 DNMT3A catalytic domain NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAM GVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV 126 Connector (L2) SSGNSNANSRGPSFSSGLVPLSLRGSH 122 DNMT3L interaction domain MGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQ RPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL 127 Connector (L1) GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE 123 Connector (L3A) + Buffer GGSGGGSA 3271 dCasX491 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 4 Buffer + Connector (L3B) RSGGSGGGSTS 3272 Suppressor subdomain 1 Human ZIM3 MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESL 3240 Human ZNF10 MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP 3239 Columba livia repressor subdomain (domain_7694) QDVVTFKDVAIYFSPEEWVRLSAGQRELYQEVMLDNYELVTSLDRESKLLYKMDPEEESCEGVPYSSADSGAPDSSSTSAC 130 Rattus norvegicus repressor subdomain ( domain_10123) ALVTFEDVAVRFTQEEWALLDPSQKILYRDVMRETYRNLTSVGINWECWDLEACFRSLGRNLRVQVVKRKCELTNSGPCAE 131 Panamanian capuchin monkey ( Cebus imitator ) suppressor subdomain ( domain_15507) SKAPITFGDLAIYFSQEEWEWLSPIQKDLYEDVMLENYRNLVSLGLSFRRPNVITLLEKGKAPWMVEPARRRRGPDSGSKV 132 Chimpanzee repressor subdomain (domain_17905) EMGLTFRDIAIEFSLEEWQCLDCAQRNLYRDVMLENYRNLVSLGIAVSKPDLITCLEQNKESQNIKRNKMVAKHPVMHSH 133 Green monkey ( Chlorocebus sabaeus ) inhibitory subdomain ( domain_20505) SQESVAFEDVAVYFTTKEWAIMVPAERALYRDVMLENYEAVAFVAVPPTSKPALVSHLEQGKESCFIRPPGVLSRSDWRAG 134 Ophiophagus hannah inhibitory subdomain ( domain_26749) STPVTFEDVVVYFTAAEWVHLTNWQRDFYQAVMMETYELVASVAGDGVPMAEDEEGGVERPVWQYIPRGKRRRKTPQPRAD 135 Giant panda ( Ailuropoda melanoleuca ) inhibitory subdomain ( domain_27604) LLTFEDVAVSFSEEEWELLDPPQKTLYNDVMQENYETVISLGLKLKNDTGNDQPISISALEMQASGSKVLRKARMKVAQKT 136 Peromyscus maniculatus bairdii suppressor subdomain ( domain_29304) VTYNDVHVDITQEEWALMDPSQRNLYKDVMVETYMNLTAIGYNWENLEVEEPCQNPLKHGRHERPHTGEKPYEYNQCGKAFAQP 137 Phyllostomus discolor suppressor subdomain ( domain_30173) VAFRDVIVDFTQEEWQQLKPAQKDLYRDVMMEIYWNLVSLDLETEGDMNEPDPEKDSWEDRTSIVVVEGLMRNGAQGYACEKAGIQGCRV 138 Buffer + NLS TSPKKKRKV 3273 LTRP4 with ADD domain Start codon + NLS + buffer sequence See above sequence 3269 Suppressor subdomain 1 Human ZIM3 See above sequence 3240 Human ZNF10 See above sequence 3239 Proto-pigeon repressor subdomain (domain_7694) See above sequence 130 Rattus norvegicus suppressor subdomain ( domain_10123) See above sequence 131 Panamanian capuchin suppressor subdomain ( domain_15507) See above sequence 132 Chimpanzee Suppressor Subdomain (domain_17905) See above sequence 133 Green Monkey Suppression Subdomain ( domain_20505) See above sequence 134 King Cobra Inhibitor Subdomain ( Domain_26749) See above sequence 135 Panda suppression subdomain ( domain_27604) See above sequence 136 Eastern deer mouse suppressor subdomain ( domain_29304) See above sequence 137 Lanceolus suppression subdomain ( domain_30173) See above sequence 138 Connector (L3A) + Buffer See above sequence 3271 Start codon + DNMT3A ADD domain See above sequence 3270 DNMT3A catalytic domain See above sequence 126 Connector (L2) See above sequence 122 DNMT3L interaction domain See above sequence 127 Connector (L1) See above sequence 123 dCasX491 See above sequence 4 Buffer + Connector (L3B) See above sequence 3272 NLS See above sequence 30 LTRP5 with ADD domain Start codon + NLS + buffer sequence See above sequence 3269 Start codon + DNMT3A ADD domain See above sequence 3270 DNMT3A catalytic domain See above sequence 126 Connector (L2) See above sequence 122 DNMT3L interaction domain See above sequence 127 Connector (L3A) See above sequence 124 Suppressor subdomain 1 Human ZIM3 See above sequence 3240 Human ZNF10 See above sequence 3239 Proto-pigeon repressor subdomain (domain_7694) See above sequence 130 Rattus norvegicus suppressor subdomain ( domain_10123) See above sequence 131 Panamanian capuchin suppressor subdomain ( domain_15507) See above sequence 132 Chimpanzee Suppressor Subdomain (domain_17905) See above sequence 133 Green Monkey Suppression Subdomain ( domain_20505) See above sequence 134 King Cobra Inhibitor Subdomain ( Domain_26749) See above sequence 135 Panda suppression subdomain ( domain_27604) See above sequence 136 Eastern deer mouse suppressor subdomain ( domain_29304) See above sequence 137 Lanceolus suppression subdomain ( domain_30173) See above sequence 138 Connector (L1) See above sequence 123 dCasX491 See above sequence 4 Buffer + Connector (L3B) See above sequence 3272 NLS See above sequence 30

Table 41 provides exemplary amino acid sequences of the components of the LTRP construct. In Table 41, the protein domain without the initiator methionine is shown. Table 41 : Exemplary protein sequences of the components of the LTRP construct Components Protein sequence SEQ ID NO DNMT3A catalytic domain (CD) 126 DNMT3L interaction domain 127 dCasX491 4 Connector 1 (L1) 123 Connector 2 (L2) 122 Connector 3A (L3A) 124 Connector 3B (L3B) 120 NLS 30 DNMT3A ADD domain 125 Example 11 : Demonstration that inclusion of a second inhibitory subdomain enhances the activity of LTRP-ADD molecules while maintaining their specificity

Experiments were conducted to determine whether incorporation of a second inhibitory subdomain would enhance the activity and specificity of LTRP molecules containing an ADD domain. This second inhibitory subdomain was positioned at the C-terminus of the LTRP5-ADD molecule previously described in Example 7, resulting in an LTRP molecule with a new configuration (described below as an LTRP6 molecule). The schematic diagram in Figure 54 shows LTRP6 molecules with configuration 6a or 6b. Molecules with configuration #6a utilize the same inhibitory subdomain at the N-terminal and C-terminal positions relative to dCasX, while molecules with configuration 6b use two different inhibitory subdomains at the two positions.

In this example, experiments were performed to assess whether incorporation of a second repressor domain at the C-terminus of the LTRP5-ADD-ZIM3 molecule to generate a LTRP6 molecule with configuration 6b would induce long-term repression of the target locus. Materials and Methods: Generation of LTRP6 constructs and plastid selection:

Plasmid constructs encoding LTRP6 molecules using the ZIM3-KRAB domain as the first inhibitory subdomain were constructed using standard molecular cloning techniques as described. Briefly, representative members of the top 95 inhibitory subdomains (Table 42) were cloned as the second inhibitory subdomain on the C-terminus of the LTRP5-ADD-ZIM3 construct generated as described in Example 7 (where the DNA and protein sequences are provided in Table 37). The LTRP5-ADD-ZIM3 molecule served as an experimental control. The plasmid also had a construct encoding the gRNA scaffold variant 174, which had a spacer targeting the endogenous B2M locus (spacer 7.165; UCCCUAUGUCCUUGCUGUUU; SEQ ID NO: 3114) or a non-targeting control. These constructs were all cloned upstream of the P2A puromycin element on the lentiviral plasmid. Transfection of HEK293T cells:

The seeded HEK293T cells were transiently transfected with 100 ng of LTRP plasmids, each of which contained a LTRP:gRNA construct encoding a LTRP6 variant containing a ZIM3-KRAB domain as the first repressor domain and an enhanced repressor domain as the second repressor domain, and a gRNA with a non-targeting spacer or a spacer targeting B2M . 24 hours after transfection, cells were selected with 1 μg/mL puromycin for three days. Cells were collected on days 4, 8, and 13 after transfection for inhibition analysis. Briefly, inhibition analysis was performed by analyzing B2M protein expression by HLA immunostaining followed by flow cytometry as described in Example 4. Results:

The effect of incorporating a second inhibitory subdomain into the LTRP5-ADD-ZIM3 molecule, thereby generating a LTRP6 construct, on increasing long-term inhibition of the target B2M locus was assessed. The results of this time course experiment are depicted in Tables 43 to 45, which show the average percentage of cells characterized as HLA negative (indicative of B2M inhibition) under each condition at 4 days, 8 days , and 13 days after transfection. The SEQ ID NOs corresponding to the domain IDs in the table below are provided in Table 42. Table 42 : List of the top 9 and top 95 most potent inhibitory subdomains Domain ID describe SEQ ID NO The first 9 inhibitory subdomains Domain_7694 Proto-pigeon repressor subdomain 130 Domain_10123 Rattus norvegicus repressor subdomain 131 Domain_15507 Panamanian capuchin monkey suppressor subdomain 132 Domain_17905 Chimpanzee repressor subdomain 133 Domain_20505 Green monkey inhibitory subdomain 134 Domain_26749 King cobra inhibitory subdomain 135 Domain_27604 Panda repressor domain 136 Domain_29304 Eastern deer mouse suppression subdomain 137 Domain_30173 Lanceolus repressor subdomain 138 The remaining inhibitory subdomains in the first 95 inhibitory subdomains Domain_737 Bonobo repressor domain 139 Domain_10331 Angolan black and white colobus monkey (Colobus angolensis palliatus) suppressor subdomain 140 Domain_10948 Angolan black-and-white colobus monkey suppressor subdomain 141 Domain_11029 Black-faced mandrill (Mandrillus leucophaeus) suppressor subdomain 142 Domain_17358 Bos indicus x Bos Taurus suppressor domain 143 Domain_17759 Felis catus repressor domain 144 Domain_18258 Sperm whale (Physeter microcephalus) repressor domain 145 Domain_19804 Northern fur seal (Callorhinus ursinus) suppression subdomain 146 Domain_221 Bonobos repress subdomain 147 Domain_881 Bonobos repress subdomain 148 Domain_2380 Orangutan repressor domain 149 Domain_2942 Gibbon repressor subdomain 150 Domain_4687 Marmoset suppression subdomain 151 Domain_4806 marmoset repressor subdomain 152 Domain_4968 marmoset repressor subdomain 153 Domain_5066 marmoset repressor subdomain 154 Domain_5290 Owl Monkey inhibitory subdomain 155 Domain_5463 Owl monkey inhibitory subdomain 156 Domain_6248 Bolivian squirrel monkey (Saimiri boliviensis boliviensis) suppressor domain 157 Domain_6445 Alligator sinensis inhibitory subdomain 158 Domain_6802 Pantherophis guttatus repressor domain 159 Domain_6807 Xenopus laevis repressor domain 160 Domain_7255 Microcaecilia unicolor inhibitory subdomain 161 Domain_8503 Mus caroli repressor domain 162 Domain_8790 Marmota monax repressor subdomain 163 Domain_8853 Syrian hamster (Mesocricetus auratus) inhibitory subdomain 164 Domain_9114 Eastern deer mouse suppression subdomain 165 Domain_9331 Bayesian deer mouse suppression subdomain 166 Domain_9538 Mus musculus inhibitory subdomain 167 Domain_9960 Octodon degus repressor domain 168 Domain_10277 Dipodomys ordii repressor domain 169 Domain_10577 Angolan black-and-white colobus monkey suppressor subdomain 170 Domain_11348 Green monkey inhibitory subdomain 171 Domain_11386 Capra hircus repressor domain 172 Domain_11486 Bos mutus repressor domain 173 Domain_11683 Nomascus leucogenys repressor domain 174 Domain_12292 Sus scrofa repressor domain 175 Domain_12452 Yangtze River Porpoise (Neophocaena asiaeorientalis asiaeorientalis) repressor domain 176 Domain_12631 Macaca fascicularis inhibitory subdomain 177 Domain_13331 Cynomolgus macaque inhibitory subdomain 178 Domain_13468 Koala (Phascolarctos cinereus) inhibitory subdomain 179 Domain_13539 Gorilla repressor subdomain 180 Domain_14659 Acinonyx jubatus repressor subdomain 181 Domain_14755 Panamanian capuchin monkey suppressor subdomain 182 Domain_15126 Common marmoset (Callithrix jacchus) suppressor subdomain 183 Domain_16444 Cheetah inhibitory subdomain 184 Domain_16688 Lipotes vexillifer repressor domain 185 Domain_16806 Capuchin monkey (Sapajus apaella) repressor domain 186 Domain_17317 Otolemur garnettii repressor domain 187 Domain_17432 Small-eared macaque inhibitory subdomain 188 Domain_18137 Gray short-tailed rat (Monodelphis domestica) suppression subdomain 189 Domain_18216 Sperm whale inhibitory subdomain 190 Domain_18563 Owl monkey inhibitory subdomain 191 Domain_19229 Alaskan sea otter (Enhydra lutris kenyoni) repressor domain 192 Domain_19460 Gray short-tailed rat inhibitory subdomain 193 Domain_19476 Owl monkey inhibitory subdomain 194 Domain_19821 Rhinopithecus roxellana inhibitory subdomain 195 Domain_19892 Polar bear (Ursus maritimus) suppression subdomain 196 Domain_19896 Ovis aries repressor subdomain 197 Domain_19949 Northern fur seal suppression subdomain 198 Domain_21247 Neovison vison repressor domain 199 Domain_21317 Pteropus vampyrus inhibitory subdomain 200 Domain_21336 Equus caballus inhibitory subdomain 201 Domain_21603 Baiji repressor domain 202 Domain_21755 Horse inhibitory subdomain 203 Domain_22153 California Sea Lion (Zalophus californianus) Suppression Subdomain 204 Domain_22270 Bonobos repress subdomain 205 Domain_23394 Vicugna pacos repressor domain 206 Domain_23723 Carlito syrichta inhibitory subdomain 207 Domain_24125 Bolivian squirrel monkey suppressor subdomain 208 Domain_24458 Iberian lynx (Lynx pardinus) repressor subdomain 209 Domain_24663 Myotis brandtii repressor domain 210 Domain_25289 Polar bear suppression subdomain 211 Domain_25379 Capuchin repressor subdomain 212 Domain_25405 Vampire bat (Desmodus rotundus) suppressor domain 213 Domain_26070 Geotrypetes seraphini repressor domain 214 Domain_26322 Caecilians 215 Domain_26732 Wild turkey (Meleagris gallopavo) repressor subdomain 216 Domain_27060 Gopherus agassizii repressor domain 217 Domain_27385 Octodon repressor domain 218 Domain_27506 yaks inhibitory subdomain 219 Domain_27811 Common marmoset suppressor domain 220 Domain_28640 Colinus virginianus repressor domain 221 Domain_28803 Gray short-tailed rat inhibitory subdomain 222 Domain_30661 Sperm whale inhibitory subdomain 223 Domain_31643 South American coral snake (Micrurus lemniscatus lemniscatus) suppressor domain 224 Table 43 : Levels of B2M inhibition mediated by LTRP constructs with various repressor domains quantified 4 days after transfection . The domain ID for each LTRP6 construct shown indicates the secondary repressor domain used and assessed Repressor construct Spacer Average HLA- negative cells % Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 6.827 0.359 3 LTRP5-ADD-ZIM3 7.165 53.833 1.159 3 LTRP6_domain_20505 7.165 69.333 0.493 3 LTRP6_domain_30173 7.165 70.267 1.115 3 LTRP6_domain_15507 7.165 71.800 0.436 3 LTRP6_domain_18137 7.165 71.900 0.600 3 LTRP6_domain_24458 7.165 73.333 0.961 3 LTRP6_domain_6445 7.165 73.567 0.666 3 LTRP6_domain_17432 7.165 74.667 0.603 3 LTRP6_domain_9960 7.165 76.000 0.100 3 LTRP6_domain_13468 7.165 76.167 0.493 3 LTRP6_domain_8853 7.165 77.400 0.173 3 LTRP6_domain_6802 7.165 77.700 0.361 3 LTRP6_domain_7694 7.165 78.300 0.361 3 LTRP6_domain_29304 7.165 78.933 0.814 3 LTRP6_domain_25379 7.165 80.033 0.569 3 LTRP6_domain_22153 7.165 80.200 1.493 3 LTRP6_domain_7255 7.165 80.567 0.551 3 LTRP6_domain_26749 7.165 81.467 0.503 3 LTRP6_domain_10123 7.165 81.833 0.289 3 Table 44 : Levels of B2M repression mediated by LTRP constructs with various repressor domains quantified 8 days after transfection . The domain ID for each LTRP6 construct shown indicates the secondary repressor domain used and assessed Repressor construct Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 2.100 0.311 2 LTRP5-ADD-ZIM3 7.165 55.000 0.300 3 LTRP6_domain_20505 7.165 72.367 1.686 3 LTRP6_domain_30173 7.165 72.733 1.069 3 LTRP6_domain_15507 7.165 74.033 0.351 3 LTRP6_domain_18137 7.165 75.100 1.253 3 LTRP6_domain_24458 7.165 76.167 0.737 3 LTRP6_domain_6445 7.165 77.600 1.249 3 LTRP6_domain_17432 7.165 78.233 0.153 3 LTRP6_domain_9960 7.165 77.600 0.346 3 LTRP6_domain_13468 7.165 79.067 0.651 3 LTRP6_domain_8853 7.165 79.333 1.150 3 LTRP6_domain_6802 7.165 78.400 0.265 3 LTRP6_domain_7694 7.165 81.433 0.416 3 LTRP6_domain_29304 7.165 80.433 0.569 3 LTRP6_domain_25379 7.165 82.167 0.351 3 LTRP6_domain_22153 7.165 81.333 1.210 3 LTRP6_domain_7255 7.165 80.933 0.404 3 LTRP6_domain_26749 7.165 85.767 0.777 3 LTRP6_domain_10123 7.165 83.000 0.625 3 Table 45 : Levels of B2M inhibition mediated by LTRP constructs with various repressor domains quantified 13 days after transfection . The domain ID for each LTRP6 construct shown indicates the secondary repressor domain used and assessed Repressor construct Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 1.587 0.670 3 LTRP5-ADD-ZIM3 7.165 50.267 0.987 3 LTRP6_domain_20505 7.165 70.133 0.945 3 LTRP6_domain_30173 7.165 70.233 0.666 3 LTRP6_domain_15507 7.165 72.067 0.252 3 LTRP6_domain_18137 7.165 72.767 1.514 3 LTRP6_domain_24458 7.165 73.967 0.404 3 LTRP6_domain_6445 7.165 75.967 0.723 3 LTRP6_domain_17432 7.165 75.667 0.351 3 LTRP6_domain_9960 7.165 74.233 0.814 3 LTRP6_domain_13468 7.165 76.767 0.764 3 LTRP6_domain_8853 7.165 75.133 1.457 3 LTRP6_domain_6802 7.165 74.700 1.136 3 LTRP6_domain_7694 7.165 78.967 0.321 3 LTRP6_domain_29304 7.165 78.233 1.332 3 LTRP6_domain_25379 7.165 78.600 0.985 3 LTRP6_domain_22153 7.165 77.533 0.839 3 LTRP6_domain_7255 7.165 77.700 0.265 3 LTRP6_domain_26749 7.165 83.800 1.127 3 LTRP6_domain_10123 7.165 79.367 0.702 3

The data in Tables 43 to 45 show that all LTRP6 constructs with B2M targeting gRNAs were able to induce higher levels of B2M inhibition by at least 13 days after transfection, and there were slight variations in the inhibitory potency depending on the inhibitory subdomain used as the second inhibitory subdomain. It is noteworthy that the use of domain_26749 (from King Cobra) as the second inhibitory subdomain caused about 83.8% B2M inhibition, which was the highest level of inhibition among the other inhibitory subdomains evaluated. In contrast, the LTRP5-ADD-ZIM3 construct only achieved an inhibition level of about 50.3%, which was about 40% lower than the inhibition level achieved by the LTRP6 construct with ZIM3 and domain_26749 inhibitory subdomains (Tables 43 to 45). The improvement in inhibition observed from these experiments can be explained by the synergistic effect of using a more potent inhibitory subdomain, the presence of the ADD domain, and the incorporation of a second inhibitory subdomain in the LTRP6 construct configuration. Example 12 below further investigates the effect of using two enhanced inhibitory subdomains on the activity and specificity of the LTRP6 construct.

These experimental results indicate that incorporation of a second repressor domain can enhance the activity of LTRP molecules containing an ADD domain. The results described herein show that incorporation of an enhancing repressor domain at the C-terminus of the LTRP5-ADD-ZIM3 molecule, generating the LTRP6 construct, improves repressor activity at the endogenous locus in human cells. Example 12 : Evaluation of the effects of the use of two repressor domains and their relative position within the LTRP6 molecule on activity and specificity

Experiments were conducted to determine whether variations of the LTRP configuration 6 (LTRP6) molecule previously described in Example 11 would result in improved long-term repression of the target locus and reduced off-target methylation. Here, the effects of using an enhancing repressor domain as the first and second repressor domains within the LTRP6 molecule and the relative positions of these repressor domains were evaluated. As shown in Figure 61, two variations of the LTRP6 configuration were tested in this example. Molecules with configuration 6a utilized the same repressor domain at the N-terminal and C-terminal positions relative to dCasX, while molecules with configuration 6b used two different repressor domains at these two positions. Materials and Methods: Generation of LTRP6 constructs and plastid selection:

Standard molecular cloning techniques were used to construct plastid constructs encoding variants of LTRP6 molecules having configuration 6a or 6b as shown in the schematic diagram of Figure 61. In this example, variants of LTRP6 molecules were generated using the following four inhibitory subdomains: domain_22153, domain_7255, domain_26749, and domain_10123. These four domains were selected because when they were incorporated as the second inhibitory subdomain in the LTRP6 configuration, they induced the highest level of B2M inhibition 4 days after transfection, as shown in Example 11 (Tables 43 to 45). Each of the four inhibitory subdomains was tested in combination relative to dCasX at the N-terminal and/or C-terminal positions, resulting in a total of 16 evaluation combinations. The coding sequences of the 16 LTRP6 variants are shown in Table 46 and their corresponding protein sequences are shown in Table 47. The plasmid also had constructs encoding gRNA scaffold variant 174 with a spacer targeting the endogenous B2M locus (spacer 7.165; UCCCUAUGUCCUUGCUGUUU; SEQ ID NO: 3114) or a non-targeting control. These constructs were all cloned upstream of the P2A puromycin element on the lentiviral plasmid. Table 46 : Coding sequences of the 16 LTRP6 variants evaluated in this example * Components Domain Combinations# domain DNA Sequence Start codon + NLS + buffer sequence ATGGCCCCAAAGAAGAAGCGGAAGGTCTCTAGA Start codon + DNMT3A ADD domain ATGGAGCGGCTGGTGTATGAGGTGCGCCAGAAGTGCAGAAACATCGAGGACATTTGTATCTCATGTGGGAGCCTCAATGTCACCCTGGAGCACCCACTCTTCATTGGAGGCATGTGCCAGAACTGTAAGAACTGCTTCTTGGAGTGTGCTTACCAGTATGACGACGATGGGTACCAGTCCTATTGCACCATCTGCTGTGGGGGG CGTGAAGTGCTCATGTGTGGGAACAACAACTGCTGCAGGTGCTTTTGTGTCGAGTGTGTGGATCTCTTGGTGGGGCCAGGAGCTGCTCAGGCAGCCATTAAGGAAGACCCCTGGAACTGCTACATGTGCGGGCATAAGGGCACCTATGGGCTGCTGCGAAGACGGGAAGACTGGCCTTCTCGACTCCAGATGTTCTTTGCCAAT DNMT3A catalytic domain AACCATGACCAGGAATTTGACCCCCCAAAGGTTTACCCACCTGTGCCAGCTGAGAAGAGGAAGCCCATCCGCGTGCTGTCTCTCTTGATGGGATTGCTACAGGGCTCCTGGTGCTGAAGGACCTGGGCATCCAAGTGGACCGCTACATCGCCTCCGAGGTGTGAGGACTCCATCACGGTGGGCATGGTGCGGCACCAGGGAAAGATCATGTACGTCGGGGAC GTCCGCAGCGTCACACAGAAGCATATCCAGGAGTGGGGCCCATTCGACCTGGTGATTGGAGGCAGTCCCTGCAACGACCTCTCCATTGTCAACCCTGCCCGCAAGGGACTTTATGAGGGTACTGGCCGCCTCTTCTTTGAGTTCTACCGCCTCCTGCATGATGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTTTGAGAATGTGGTGGCCATGG GCGTTAGTGACAAGAGGGACATCTCGCGATTTCTTGAGTCTAACCCCGTGATGATTGACGCCAAAGAAGTGTCTGCTGCACACAGGGCCCGTTACTTCTGGGGTAACCTTCCTGGCATGAACAGGCCTTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAAGAGTGTCTGGAGCACGGCAGAATAGCCAAGTTCAGCAAAGTGAGGACCATTACCACCAGGTC AAACTCTATAAAGCAGGGCAAAGACCAGCATTTCCCCGTCTTCATGAACGAAGGAGGACATCCTGTGGTGCACTGAAATGGAAAGGGTGTTTGGCTTCCCCGTCCACTACACAGACGTGTCCAACATGAGCCGCTTGGCGAGGCAGAGACTGCTGGGCCGGTCGTGGAGCGTGCCGGTCATCCGCCACCTCTTCGCTCCGCTGAAGGAATATTTTGCTTGTGTG Connector 2 (L2) TCTAGCGGCAATAGTAACGCTAACAGCCGCGGGCCGAGCTTCAGCAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAGCCAT DNMT3L interaction domain ATGGGCCCTATGGAGATATACAAGACAGTGTCTGCATGGAAGAGACAGCCAGTGCGGGTACTGAGCCTCTTCAGAAACATCGACAAGGTACTAAAGAGTTTGGGCTTCTTGGAAAGCGGTTCTGGTTCTGGGGGAGGAACGCTGAAGTACGTGGAAGATGT CACAAATGTCGTGAGGAGGGACGTGGAGAAATGGGGCCCCTTTGACCTGGTGTACGGCTCGACGCAGCCCCTAGGCAGCTCTTGTGATCGCTGTCCCGGCTGGTACATGTTCCAGTTCCACCGGATTCTGCAGTATGCGCTGCCTCGCCAGGAGAGTCAGC GGCCCTTCTTCTGGATATTCATGGACAATCTGCTGCTGACTGAGGATGACCAAGAGACAACTACCCGCTTCCTTCAGACAGAGGCTGTGACCCTCCAGGATGTCCGTGGCAGAGACTACCAGAATGCTATGCGGGTGTGGAGCAACATTCCAGGGCTGAAG AGCAAGCATGCGCCCCTGACCCCAAAGGAAGAAGAGTATCTGCAAGCCCAAGTCAGAAGCAGGAGCAAGCTGGACGCCCCGAAAGTTGACCTCCTGGTGAAGAACTGCCTTCTCCCGCTGAGAGAGTACTTCAAGTATTTTTCTCAAAACTCACTTCCTCTT Connector 3A (L3A) GGCGGTTCCGGCGGAGGA N-terminal inhibitory subdomain Combination 1, 5, 9 and 13 Domain_22153 GTTCGTAGCCTGTTACTTTTGAAGATGTTGCTGTTACTTTTACTCAAGAAGAATGGGGGCAATTAGATCCTGCTCAACGTACTTTATATCAAGAAGTTATGTTAGAGACTTGTGGTTTATTAGTTTCTTTAGGTTGTCCTTTACCTAAACCTGAATTATTTTATCCTTTAGATCATTCTCCTGAATTACAAACTTTAAAACGTGGTTTATCTCCTAATTCTTGTCCTGGTGATTCTACTAAA Combination 2, 6, 10 and 14 Domain_7255 ACTCAAGTTCCTGTTACCTTTGAAGATGTTGCTGTTTATTTTTGTGAAGGTGAATGGGAAACTTTAGCTGAATGGCAAAAAGAATTATATCGTGAAACTATGAAAGAATTATGAAACTTTAACTTCTTTAGGTTTTTCTTCTAAAAAACCCTTCTTTAATTTCTAAAATAGAACGTGAAGAAGATCCTTGTGTGCGTGATAAACAAGATTCTCGTGATCGTCGTCGTTTACGTTCTTGTTGG Combination 3, 7, 11 and 15 Domain_26749 TCTACTCCTGTTACTTTTGAAGATGTTGTTGTTTATTTTACTGCTGCTGAATGGGTTCATTTAACTAATTGGCAACGTGATTTTTATCAAGCTGTTATGATGGAAACTTATGAATTAGTTGCTTCTGTTGCTGGTGATGGTGTTCCTATGGCTGAAGATGAAGAAGGTGGTGTTGAACGTCCTGTTTGGCAATATATTCCTCGTGGTAAACGTCGTCGTAAAACTCCTCAACCTCGTGCTGAT Combination 4, 8, 12 and 16 Domain_10123 GCTTTAGTTACTTTTGAAGATGTTGCTGTTCGTTTTACTCAAGAAGAATGGGCTTTATTAGATCCTTCTCAAAAAATTTTATATCGTGATGTTATGCGTGAGACTTATCGTAATTTAACTTCTGTTGGTATATTAATTGGGAATGTTGGGATTTAGAAGCTTGTTTTCGTTCTTTAGGTCGTAATTTACGTGTTCAAGTCGTTAAACGTAAATGTGAATTAACTAATTCTGGTCCTTGTGCTGAA Connector 1 (L1) GGAGGGCCGAGCTCTGGCGCACCCCCACCAAGTGGAGGGTCTCCTGCCGGGTCCCCCAACATCTACTGAAGAAGGCACCAGCGAATCCGCAACGCCCGAGTCAGGCCCTGGTACCTCCACAGAACCATCTGAAGGTAGTGCGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCACCGAAGAAGGCACGTCAACCGAACCAAGTGAAGGATCTGCCCCTGGGACCAGCACTGAACCATCTGAG dX dCasX491 CAAGAGATCAAGAGAATCAACAAGATCAGAAGGAGACTGGTCAAGGACAGCAACACAAAGAAGGCCGGCAAGACAGGCCCCATGAAAACCCTGCTCGTCAGAGTGATGACCCCTGACCTGAGAGAGCGGCTGGAAAACCTGAGAAAGAAGCCCGAGAACATCCCTCAGCCTATCAGCAACACC AGCAGGGCCAACCTGAACAAGCTGCTGACCGACTACACCGAGATGAAGAAAGCCATCCTGCACGTGTACTGGGAAGAGTTCCAGAAAGACCCCGTGGGCCTGATGAGCAGAGTTGCTCAGCCTGCCAGCAAGAAGATCGACCAGAACAAGCTGAAGCCCGAGATGGACGAGAAGGGCAATCTG ACCACAGCCGGCTTTGCCTGCTCTCAGTGTGGCCAGCCTCTGTTCGTGTACAAGCTGGAACAGGTGTCCGAGAAAGGCAAGGCCTACACCAACTTCGGCAGATGTAACGTGGCCGAGCACGAGAAGCTGATTCTGCTGGCCCAGCTGAAACCTGAGAAGGACTCTGATGAGGCCGTGACC TACAGCCTGGGCAAGTTTGGACAGAGAGCCCTGGACTTCTACAGCATCCACGTGACCAAAGAAAGCACACACCCCGTGAAGCCCCTGGCTCAGATCGCCGGCAATAGATACGCCTCTGGACCTGTGGGCAAAGCCCTGTCCGATGCCTGCATGGGAACAATCGCCAGCTTCCTGAGCAAGTAC CAGGACATCATCGAGCACCAGAAGGTGGTCAAGGGCAACCAGAAGAGACTGGAAAGCCTGAGGGAGCTGGCCGGCAAAGAGAACCTGGAATACCCCAGCGTGACCCTGCCTCCTCAGCCTCACACAAAAGAAGGCGTGGACGCCTACAACGAAGTGATCGCCAGAGTGAGAATGTGGGTC AACCTGAACCTGTGGCAGAAGCTGAAACTGTCCAGGGACGACGCCAAGCCTCTGCTGAGACTGAAGGGCTTCCCTAGCTTCCCTCTGGTGGAAAGACAGGCCAATGAAGTGGATTGGTGGGACATGGTCTGCAACGTGAAGAAGCTGATCAACGAGAAGAAAGAGGATGGCAAGGTTTTCTGG CAGAACCTGGCCGGCTACAAGAGACAAGAAGCCCTGAGGCCTTACCTGAGCAGCGAAGAGGACCGGAAGAAGGGCAAGAAGTTCGCCAGATACCAGCTGGGCGACCTGCTGCTGCACCTGGAAAAGAAGCACGGCGAGGACTGGGGCAAAGTGTACGATGAGGCCTGGGAGAGAATCGACAAG AAGGTGGAAGGCCTGAGCAAGCACATTAAGCTGGAAGAGGAAAGAAGGAGCGAGGACGCCCAATCTAAAGCCGCTCTGACCGATTGGCTGAGAGCCAAGGCCAGCTTTGTGATCGAGGGCCTGAAAGAGGCCGACAAGGACGAGTTCTGCAGATGCGAGCTGAAGCTGCAGAAGTGGTACGGCG ATCTGAGAGGCAAGCCCTTCGCCATTGAGGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCAGTACAACTGCGCCTTCATTTGGCAGAAAGACGGCGTCAAGAAACTGAACCTGTACCTGATCATCAATTACTTCAAAGGCGGCAAGCTGCGGTTCAAGAAGATCAAACCCGAGG CCTTCGAGGCTAACAGATTCTACACCGTGATCAACAAAAAGTCCGGCGAGATCGTGCCCATGGAAGTGAACTTCAACTTCGACGACCCCAACCTGATTATCCTGCCTCTGGCCTTCGGCAAGAGACAGGGCAGAGAGTTCATCTGGAACGATCTGCTGAGCCTGGAAACCGGCTCTCTGAAGC TGGCCAATGGCAGAGTGATCGAGAAAACCCTGTACAACAGGAGAACCAGACAGGACGAGCCTGCTCTGTTTGTGGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGCAACATCAAGCCCATGAACCTGATCGGCGTGGCCCGGGGCGAGAATATCCCTGCTGTGATCGCCCTGACAG ACCCTGAAGGATGCCCACTGAGCAGATTCAAGGACTCCCTGGGCAACCCTACACACATCCTGAGAATCGGCGAGAGCTACAAAGAGAAGCAGAGGACAATCCAGGCCAAGAAAGAGGTGGAACAGAGAAGAGCCGGCGGATACTCTAGGAAGTACGCCAGCAAGGCCAAGAATCTGGCCGACGA CATGGTCCGAAACACCGCCAGAGATCTGCTGTACTACGCCGTGACACAGGACGCCATGCTGATCTTCGCGAATCTGAGCAGAGGCTTCGGCCGGCAGGGCAAGAGAACCTTTATGGCCGAGAGGCAGTACACCAGAATGGAAGATTGGCTCACAGCTAAACTGGCCTACGAGGGACTGAGCAA GACCTACCTGTCCAAAACACTGGCCCAGTATACCTCCAAGACCTGCAGCAATTGCGGCTTCACCATCACCAGCGCCGACTACGACAGAGTGCTGGAAAAGCTCAAGAAAACCGCCACCGGCTGGATGACCACCATCAACGGCAAAGAGCTGAAGGTTGAGGGCCAGATCACCTACTACAACAG GTACAAGAGGCAGAACGTCGTGAAGGATCTGAGCGTGGAACTGGACAGACTGAGCGAAGAGAGCGTGAACAACGACATCAGCAGCTGGACAAAGGGCAGATCAGGCGAGGCTCTGAGCCTGCTGAAGAAGAGGTTTAGCCACAGACCTGTGCAAGAGAAGTTCGTGTGCCTGAACTGCGGCTT CGAGACACACGCCGCTGAACAGGCTGCCCTGAACATTGCCAGAAGCTGGCTGTTCCTGAGAAGCCAAGAGTACAAGAAGTACCAGACCAACAAGACCACCGGCAACACCGACAAGAGGGCCTTTGTGGAAAACCTGGCAGAGCTTCTACAGAAAAAAGCTGAAAGAAGTCTGGAAGCCCGCCGTG Buffer sequence + Linker 3B (L3B) + Buffer sequence CGATCGGGCGGTTCCGGCGGAGGTTCCACTAGT C-terminal inhibitory domain Combination 1 to 4 Domain_7255 ACTCAAGTTCCTGTTACCTTTGAAGATGTTGCTGTTTATTTTTGTGAAGGTGAATGGGAAACTTTAGCTGAATGGCAAAAAGAATTATATCGTGAAACTATGAAAGAATTATGAAACTTTAACTTCTTTAGGTTTTTCTTCTAAAAAACCCTTCTTTAATTTCTAAAATAGAACGTGAAGAAGATCCTTGTGTGCGTGATAAACAAGATTCTCGTGATCGTCGTCGTTTACGTTCTTGTTGG Combination 5 to 8 Domain_22153 GTTCGTAGCCTGTTACTTTTGAAGATGTTGCTGTTACTTTTACTCAAGAAGAATGGGGGCAATTAGATCCTGCTCAACGTACTTTATATCAAGAAGTTATGTTAGAGACTTGTGGTTTATTAGTTTCTTTAGGTTGTCCTTTACCTAAACCTGAATTATTTTATCCTTTAGATCATTCTCCTGAATTACAAACTTTAAAACGTGGTTTATCTCCTAATTCTTGTCCTGGTGATTCTACTAAA Combination 9 to 12 Domain_10123 GCTTTAGTTACTTTTGAAGATGTTGCTGTTCGTTTTACTCAAGAAGAATGGGCTTTATTAGATCCTTCTCAAAAAATTTTATATCGTGATGTTATGCGTGAGACTTATCGTAATTTAACTTCTGTTGGTATATTAATTGGGAATGTTGGGATTTAGAAGCTTGTTTTCGTTCTTTAGGTCGTAATTTACGTGTTCAAGTCGTTAAACGTAAATGTGAATTAACTAATTCTGGTCCTTGTGCTGAA Combination 13 to 16 Domain_26749 TCTACTCCTGTTACTTTTGAAGATGTTGTTGTTTATTTTACTGCTGCTGAATGGGTTCATTTAACTAATTGGCAACGTGATTTTTATCAAGCTGTTATGATGGAAACTTATGAATTAGTTGCTTCTGTTGCTGGTGATGGTGTTCCTATGGCTGAAGATGAAGAAGGTGGTGTTGAACGTCCTGTTTGGCAATATATTCCTCGTGGTAAACGTCGTCGTAAAACTCCTCAACCTCGTGCTGAT Linker 4v1 (L4v1) GGAAGTGGGAGC NLS CCCAAGAAAAAGAGAAAAGTG * The components are listed in 5' to 3' order within the construct Table 47 : Full-length protein sequences of the 16 LTRP6 variants evaluated in this example Components Domain Combinations# domain AA sequence Start codon + NLS + buffer sequence MAPKKKRKVSR Start codon + DNMT3A ADD domain MERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLLRRREDWPSRLQMFFAN DNMT3A catalytic domain NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAM GVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV Connector 2 (L2) SSGNSNANSRGPSFSSGLVPLSLRGSH DNMT3L interaction domain MGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQ RPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL Connector 3A (L3A) GGSGGG N-terminal inhibitory subdomain Combination 1, 5, 9 and 13 Domain_22153 VRMPVTFEDVAVTFTQEEWGQLDPAQRTLYQEVMLETCGLLVSLGCPLPKPELFYPLDHSPELQTLKRGLSPNSSCPGDSTK Combination 2, 6, 10 and 14 Domain_7255 TQVPVTFEDVAVYFCEGEWETLAEWQKELYRETMKENYETLTSLGFSSKKPSLISKIEREEDPCVRDKQDSRDRRRLRSCW Combination 3, 7, 11 and 15 Domain_26749 STPVTFEDVVVYFTAAEWVHLTNWQRDFYQAVMMETYELVASVAGDGVPMAEDEEGGVERPVWQYIPRGKRRRKTPQPRAD Combination 4, 8, 12 and 16 Domain_10123 ALVTFEDVAVRFTQEEWALLDPSQKILYRDVMRETYRNLTSVGINWECWDLEACFRSLGRNLRVQVVKRKCELTNSGPCAE Connector 1 (L1) GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE dX dCasX491 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNL TTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFW QNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYG DLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLAD DMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV Buffer sequence + Linker 3B (L3B) + Buffer sequence RSGGSGGGSTS C-terminal inhibitory domain Combination 1 to 4 Domain_7255 TQVPVTFEDVAVYFCEGEWETLAEWQKELYRETMKENYETLTSLGFSSKKPSLISKIEREEDPCVRDKQDSRDRRRLRSCW Combination 5 to 8 Domain_22153 VRMPVTFEDVAVTFTQEEWGQLDPAQRTLYQEVMLETCGLLVSLGCPLPKPELFYPLDHSPELQTLKRGLSPNSSCPGDSTK Combination 9 to 12 Domain_10123 ALVTFEDVAVRFTQEEWALLDPSQKILYRDVMRETYRNLTSVGINWECWDLEACFRSLGRNLRVQVVKRKCELTNSGPCAE Combination 13 to 16 Domain_26749 STPVTFEDVVVYFTAAEWVHLTNWQRDFYQAVMMETYELVASVAGDGVPMAEDEEGGVERPVWQYIPRGKRRRKTPQPRAD Linker 4v1 (L4v1) GSGS NLS PKKKRKV

Transfection of HEK293T cells was performed in a manner similar to that described in Example 11. In brief, HEK293T cells were transiently transfected with plasmids encoding LTRP6 variants (listed in Table 46) and gRNAs with spacers targeting B2M . 24 hours after transfection, cells were selected for five days with puromycin. Cells were collected at 8, 12, 20, 27, 44, 59, 74, and 104 days after transfection to determine the level of B2M inhibition. LTRP5-ZIM3 molecules and LTRP5-ADD-ZIM3 molecules served as experimental controls. In addition, for comparison, this experiment also included LTRP6 variants (also referred to as LTRP6 with double ZIM3) using the ZIM3 domain as the first and second inhibitory subdomains for comparison.

Bisulfite sequencing was also performed to assess off-target methylation at the VEGFA locus, which was performed as described in Example 5. Briefly, five days after transfection, HEK293T cells transiently transfected with LTRP6 variant plasmids with B2M- targeting gRNA or non-targeting gRNA were collected to extract gDNA for bisulfite sequencing. The following controls were also included: 1) CasX 491 paired with B2M spacer 7.37 (GGCCGAGAUGUCUCGCUCCG; SEQ ID NO: 3137); 2) dCas9-ZNF10-DNMT3A/3L with its corresponding B2M spacer; 3) LTRP1 with non-targeting or B2M spacer 7.165 (described in Figure 28); 4) LTRP5-ZIM5 with B2M spacer 7.165; and 5) LTRP5-ADD-ZIM3 with non-targeting or B2M spacer 7.165. Results:

The effects of using the enhancing repressor domain as the first and second repressor domains and their relative positioning within the LTRP6 molecule were assessed. The results of the time course experiments evaluating the effects on long-term repression of the target B2M locus are depicted in Tables 48 to 55. Overall, the data indicate that incorporation of the second repressor domain into the C-terminus of the LTRP5-ADD molecule (generating a LTRP6 configuration) substantially improves long-lasting B2M repression, reproducing the findings observed in Example 11. Addition of a ZIM3 domain to the C-terminus of LTRP5-ADD-ZIM3, resulting in LTRP6-bi-ZIM3, enhanced long-term inhibition when compared to the level of B2M inhibition achieved by the LTRP5-ADD-ZIM3 construct at least until 59 days post-transfection, but this improvement in long-term inhibition did not persist until day 104 (Tables 48-55). The enhanced inhibition observed with the second ZIM3 domain further supports that the observed improvement in activity of the LTRP6 configuration is due to the presence of the second inhibitory subdomain C-terminally linked to dCasX. Most of the other LTRP6 constructs with two enhanced inhibitory subdomains further improved inhibitory activity compared to LTRP6-bi-ZIM3, even until 104 days post-transfection. Notably, LTRP6 molecules containing domain-26749 (at the N-terminus and/or C-terminus) were the best performing molecules in inducing persistent B2M inhibition (>90%) until day 104, with LTRP6-double-26749 (Configuration 6a) showing the highest level of inhibition and LTRP6-10123-26749 (Configuration 6b) exhibiting the second highest inhibitory activity (Tables 48 to 55). It is worth noting that in some cases, localization of the inhibitory subdomain to the N-terminus of dCasX also affected the activity of the molecule compared to localization to the C-terminus. For example, the combination of domain_7255 located on the N-terminus of dCasX and domain_22153 located on the C-terminus caused about 58% inhibition; however, when domain_22153 was located on the N-terminus and domain_7255 was located on the C-terminus, the inhibition level increased to 91% (Tables 48 to 55). Table 48 : Levels of B2M inhibition mediated by LTRP constructs with various inhibitory subdomains quantified 8 days after transfection . The order of the domain IDs displayed for each LTRP6 construct specifies the first inhibitory subdomain and the second inhibitory subdomain at the N- terminal and C- terminal positions, respectively. Repressor construct Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 8.437 2.483 3 LTRP5-ZIM3 7.165 61.133 2.003 3 LTRP5-ADD-ZIM3 7.165 74.967 1.443 3 LTRP6-Double-ZIM3 7.165 89.833 0.416 3 LTRP6-22153-7255 7.165 92.200 0.361 3 LTRP6-Double-7255 7.165 91.267 0.321 3 LTRP6-26749-7255 7.165 92.567 0.208 3 LTRP6-10123-7255 7.165 92.100 0.529 3 LTRP6-Double-22153 7.165 88.700 0.600 3 LTRP6-7255-22153 7.165 91.500 0.100 3 LTRP6-26749-22153 7.165 93.033 0.737 3 LTRP6-10123-22153 7.165 91.967 0.702 3 LTRP6-22153-10123 7.165 91.767 1.405 3 LTRP6-7255-10123 7.165 90.800 2.128 3 LTRP6-26749-10123 7.165 94.000 1.082 3 LTRP6-Double-10123 7.165 93.100 1.136 3 LTRP6-22153-26749 7.165 92.167 1.474 3 LTRP6-7255-26749 7.165 91.567 1.365 3 LTRP6-Double-26749 7.165 92.100 1.044 3 LTRP6-10123-26749 7.165 92.367 0.802 3 Table 49 : Levels of B2M inhibition mediated by LTRP constructs with various inhibitory subdomains quantified 12 days after transfection . The order of domain IDs shown for each LTRP6 construct specifies the first and second inhibitory subdomains at the N- terminal and C- terminal positions, respectively. Repressor construct Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 1.970 0.442 3 LTRP5-ZIM3 7.165 50.433 1.250 3 LTRP5-ADD-ZIM3 7.165 75.300 1.997 3 LTRP6-Double-ZIM3 7.165 90.067 0.404 3 LTRP6-22153-7255 7.165 93.567 0.493 3 LTRP6-Double-7255 7.165 90.400 0.173 3 LTRP6-26749-7255 7.165 94.367 0.306 3 LTRP6-10123-7255 7.165 93.300 0.608 3 LTRP6-Double-22153 7.165 89.067 0.924 3 LTRP6-7255-22153 7.165 90.733 0.058 3 LTRP6-26749-22153 7.165 95.267 0.709 3 LTRP6-10123-22153 7.165 94.500 0.608 3 LTRP6-22153-10123 7.165 93.400 0.100 3 LTRP6-7255-10123 7.165 89.667 1.210 3 LTRP6-26749-10123 7.165 95.700 0.200 3 LTRP6-Double-10123 7.165 94.033 0.289 3 LTRP6-22153-26749 7.165 95.067 0.651 3 LTRP6-7255-26749 7.165 95.300 0.866 3 LTRP6-Double-26749 7.165 95.567 0.404 3 LTRP6-10123-26749 7.165 95.400 0.608 3 Table 50 : Levels of B2M inhibition mediated by LTRP constructs with various inhibitory subdomains quantified 20 days after transfection . The order of domain IDs shown for each LTRP6 construct specifies the first and second inhibitory subdomains at the N- terminal and C- terminal positions, respectively. Repressor construct Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 6.060 3.841 3 LTRP5-ZIM3 7.165 45.233 3.958 3 LTRP5-ADD-ZIM3 7.165 71.700 2.107 3 LTRP6-Double-ZIM3 7.165 89.767 1.124 3 LTRP6-22153-7255 7.165 92.433 0.987 3 LTRP6-Double-7255 7.165 89.233 1.528 3 LTRP6-26749-7255 7.165 93.200 0.400 3 LTRP6-10123-7255 7.165 92.133 0.929 3 LTRP6-Double-22153 7.165 87.700 0.872 3 LTRP6-7255-22153 7.165 88.633 0.814 3 LTRP6-26749-22153 7.165 94.767 0.850 3 LTRP6-10123-22153 7.165 93.400 0.794 3 LTRP6-22153-10123 7.165 91.967 1.274 3 LTRP6-7255-10123 7.165 87.233 1.960 3 LTRP6-26749-10123 7.165 94.767 0.208 3 LTRP6-Double-10123 7.165 92.367 0.404 3 LTRP6-22153-26749 7.165 94.400 1.015 3 LTRP6-7255-26749 7.165 93.667 1.069 3 LTRP6-Double-26749 7.165 95.367 0.681 3 LTRP6-10123-26749 7.165 94.867 0.907 3 Table 51 : Levels of B2M inhibition mediated by LTRP constructs with various inhibitory subdomains quantified 27 days after transfection . The order of domain IDs shown for each LTRP6 construct specifies the first and second inhibitory subdomains at the N- terminal and C- terminal positions, respectively. Repressor construct Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 2.600 1.006 3 LTRP5-ZIM3 7.165 36.733 2.608 3 LTRP5-ADD-ZIM3 7.165 69.100 1.900 3 LTRP6-Double-ZIM3 7.165 87.200 1.300 3 LTRP6-22153-7255 7.165 91.300 1.179 3 LTRP6-Double-7255 7.165 87.233 1.250 3 LTRP6-26749-7255 7.165 92.367 0.551 3 LTRP6-10123-7255 7.165 90.733 1.518 3 LTRP6-Double-22153 7.165 85.567 1.007 3 LTRP6-7255-22153 7.165 86.433 1.290 3 LTRP6-26749-22153 7.165 94.067 0.681 3 LTRP6-10123-22153 7.165 92.467 1.002 3 LTRP6-22153-10123 7.165 90.900 1.646 3 LTRP6-7255-10123 7.165 85.200 1.411 3 LTRP6-26749-10123 7.165 93.567 0.643 3 LTRP6-Double-10123 7.165 91.033 0.058 3 LTRP6-22153-26749 7.165 93.900 0.693 3 LTRP6-7255-26749 7.165 92.933 1.474 3 LTRP6-Double-26749 7.165 95.300 0.557 3 LTRP6-10123-26749 7.165 94.133 1.528 3 Table 52 : Levels of B2M inhibition mediated by LTRP constructs with various repressor domains quantified 44 days after transfection . The order of domain IDs shown for each LTRP6 construct specifies the first and second repressor domains at the N- terminal and C- terminal positions, respectively. Repressor construct Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 1.307 0.070 3 LTRP5-ZIM3 7.165 26.533 3.232 3 LTRP5-ADD-ZIM3 7.165 63.700 1.345 3 LTRP6-Double-ZIM3 7.165 82.600 1.929 3 LTRP6-22153-7255 7.165 88.933 4.110 3 LTRP6-Double-7255 7.165 83.233 2.610 3 LTRP6-26749-7255 7.165 88.833 1.604 3 LTRP6-10123-7255 7.165 86.567 1.909 3 LTRP6-Double-22153 7.165 79.867 0.153 3 LTRP6-7255-22153 7.165 80.033 3.807 3 LTRP6-26749-22153 7.165 91.933 2.159 3 LTRP6-10123-22153 7.165 89.567 0.929 3 LTRP6-22153-10123 7.165 86.900 3.119 3 LTRP6-7255-10123 7.165 77.433 2.031 3 LTRP6-26749-10123 7.165 91.133 0.611 3 LTRP6-Double-10123 7.165 87.033 0.231 3 LTRP6-22153-26749 7.165 93.000 2.100 3 LTRP6-7255-26749 7.165 89.367 3.758 3 LTRP6-Double-26749 7.165 96.167 0.737 3 LTRP6-10123-26749 7.165 94.050 0.919 2 Table 53 : Levels of B2M inhibition mediated by LTRP constructs with various inhibitory subdomains quantified 59 days after transfection . The order of domain IDs shown for each LTRP6 construct specifies the first and second inhibitory subdomains at the N- terminal and C- terminal positions, respectively. Repressor construct Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 0.947 0.012 3 LTRP5-ZIM3 7.165 19.667 2.627 3 LTRP5-ADD-ZIM3 7.165 60.533 1.801 3 LTRP6-Double-ZIM3 7.165 76.567 4.274 3 LTRP6-22153-7255 7.165 87.933 6.385 3 LTRP6-Double-7255 7.165 84.100 2.211 3 LTRP6-26749-7255 7.165 85.733 1.704 3 LTRP6-10123-7255 7.165 83.267 3.868 3 LTRP6-Double-22153 7.165 74.500 0.608 3 LTRP6-7255-22153 7.165 74.133 5.463 3 LTRP6-26749-22153 7.165 89.833 3.691 3 LTRP6-10123-22153 7.165 89.200 1.825 3 LTRP6-22153-10123 7.165 84.733 3.581 3 LTRP6-7255-10123 7.165 71.667 5.980 3 LTRP6-26749-10123 7.165 90.567 0.808 3 LTRP6-Double-10123 7.165 83.167 1.026 3 LTRP6-22153-26749 7.165 93.467 2.301 3 LTRP6-7255-26749 7.165 86.900 6.077 3 LTRP6-Double-26749 7.165 96.833 0.874 3 LTRP6-10123-26749 7.165 93.450 1.202 2 Table 54 : Levels of B2M inhibition mediated by LTRP constructs with various inhibitory subdomains quantified 74 days after transfection . The order of domain IDs shown for each LTRP6 construct specifies the first and second inhibitory subdomains at the N- terminal and C- terminal positions, respectively. Repressor construct Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 2.407 1.086 3 LTRP5-ZIM3 7.165 21.033 9.890 3 LTRP5-ADD-ZIM3 7.165 61.167 5.807 3 LTRP6-Double-ZIM3 7.165 68.133 7.305 3 LTRP6-22153-7255 7.165 90.033 6.144 3 LTRP6-Double-7255 7.165 87.833 2.219 3 LTRP6-26749-7255 7.165 84.700 1.539 3 LTRP6-10123-7255 7.165 83.833 3.859 3 LTRP6-Double-22153 7.165 73.000 0.721 3 LTRP6-7255-22153 7.165 71.267 7.343 3 LTRP6-26749-22153 7.165 92.533 3.062 3 LTRP6-10123-22153 7.165 92.133 2.542 3 LTRP6-22153-10123 7.165 87.533 1.069 3 LTRP6-7255-10123 7.165 69.533 5.265 3 LTRP6-26749-10123 7.165 92.667 0.833 3 LTRP6-Double-10123 7.165 80.133 2.458 3 LTRP6-22153-26749 7.165 92.433 2.804 3 LTRP6-7255-26749 7.165 87.800 8.143 3 LTRP6-Double-26749 7.165 97.133 0.643 3 LTRP6-10123-26749 7.165 94.067 1.686 3 Table 55 : Levels of B2M inhibition mediated by LTRP constructs with various inhibitory subdomains quantified 104 days after transfection . The order of domain IDs shown for each LTRP6 construct specifies the first and second inhibitory subdomains at the N- terminal and C- terminal positions, respectively. Repressor construct Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5-ADD-ZIM3 NT 0.610 0.314 3 LTRP5-ZIM3 7.165 14.270 12.499 3 LTRP5-ADD-ZIM3 7.165 57.533 7.419 3 LTRP6-Double-ZIM3 7.165 43.467 9.843 3 LTRP6-22153-7255 7.165 90.900 1.929 3 LTRP6-Double-7255 7.165 93.367 0.603 3 LTRP6-26749-7255 7.165 84.000 1.044 3 LTRP6-10123-7255 7.165 77.700 16.664 3 LTRP6-Double-22153 7.165 69.567 6.336 3 LTRP6-7255-22153 7.165 57.567 12.667 3 LTRP6-26749-22153 7.165 87.867 4.277 3 LTRP6-10123-22153 7.165 88.600 7.134 3 LTRP6-22153-10123 7.165 84.600 0.624 3 LTRP6-7255-10123 7.165 78.033 4.922 3 LTRP6-26749-10123 7.165 94.400 1.375 3 LTRP6-Double-10123 7.165 79.100 5.216 3 LTRP6-22153-26749 7.165 92.067 5.595 3 LTRP6-7255-26749 7.165 90.067 6.274 3 LTRP6-Double-26749 7.165 98.267 0.929 3 LTRP6-10123-26749 7.165 94.633 1.779 3

Five days after transfection, bisulfite sequencing was performed to assess the extent of off-target CpG methylation at the VEGFA locus mediated by the 16 LTRP6 molecules, and the results are shown in Table 56. The data indicate that most LTRP6 constructs exhibit relatively low levels of off-target methylation, especially when compared to the levels achieved with constructs encoding dCas9-ZNF10-DNMT3A/3L, LTRP1, and LTRP5-ZIM3 controls. In fact, LTRP6 constructs containing domain_26749 (configuration 6a or 6b) at either or both positions exhibited off-target methylation similar to LTRP5-ADD-ZIM3 and LTRP6-bi-ZIM3 molecules (Table 56). LTRP6-bi-26749 and LTRP6-10123-26749 exhibited the highest and second highest inhibitory activities, respectively (Tables 48 to 55), inducing off-target methylation levels of approximately 5.15% and approximately 3.84%, respectively. Table 56 : Levels of off-target CpG methylation at the VEGFA locus mediated by LTRP6 constructs with various inhibitory subdomains quantified at day 5 after transfection . The order of the domain IDs of each LTRP6 construct displayed specifies the first and second inhibitory subdomains at the N- terminal and C- terminal positions, respectively . Constructs encoding CasX , dCas9-ZNF10-DNMT3A/3L , LTRP1 , LTRP5-ZIM3 , and LTRP5-ADD-ZIM3 were also included as controls Repressor construct Spacer Average CpG methylation % Standard error CasX 491 7.37 0.851 0.160 dCas9-ZNF10-DNMT3A/3L 7.148 34.220 2.356 LTRP1 NT 16.900 1.346 LTRP1 7.165 30.880 1.829 LTRP5-ADD-ZIM3 NT 6.887 0.403 LTRP5-ZIM5 7.165 14.690 1.338 LTRP5-ADD-ZIM3 7.165 4.841 0.508 LTRP6-Double-ZIM3 7.165 5.782 0.565 LTRP6-7255-22153 7.165 3.988 0.479 LTRP6-Double-7255 7.165 11.330 0.993 LTRP6-7255-26749 7.165 5.719 0.737 LTRP6-7255-10123 7.165 5.796 0.635 LTRP6-22153-26749 7.165 4.789 0.492 LTRP6-22153-10123 7.165 1.244 0.422 LTRP6-10123-22153 7.165 6.607 0.757 LTRP6-10123-7255 7.165 16.530 1.417 LTRP6-10123-26749 7.165 4.810 0.523 LTRP6-Double-10123 7.165 5.729 0.699 LTRP6-26749-22153 7.165 5.401 0.565 LTRP6-26749-7255 7.165 4.608 0.582 LTRP6-Double-22153 7.165 3.522 0.537 LTRP6-22153-7255 7.165 4.388 0.669 LTRP6-Double-26749 7.165 5.153 0.457 LTRP6-26749-10123 7.165 3.836 0.482

The experimental results show that incorporating a second repressor domain into the LTRP molecule to produce the LTRP6 configuration will improve persistent repression, and this improvement is further enhanced by using an enhancing repressor domain at the N-terminal and C-terminal positions relative to dCasX. In addition, the data show that the use of some but not all new repressor domains can induce very persistent repression of the target locus, and their relative positioning within the LTRP molecule can also affect activity. The findings from these experiments also show that incorporating a second repressor domain into the LTRP-ADD molecule will maintain molecular specificity while improving activity. Example 13 : Optimizing linker sequences to increase overall LTRP activity

Experiments were performed to show that the inhibitory activity of LTRP5 constructs can be increased by optimizing the linker sequences at two positions: 1) between the DNMT3L interaction domain and the inhibitory domain; and 2) between the inhibitory domain and the catalytically inactive CasX. These linker positions correspond to "L3A" and "L1" in the schematic diagram of LTRP5 configuration in Figure 55, respectively. Here, experiments were performed to screen and identify new linker combinations that would improve LTRP activity. Materials and Methods:

An entry vector with restriction enzyme sites between the DNMT3L interaction domain and the inhibitory domain and between the inhibitory domain and the catalytically inactive CasX oriented in the LTRP5 configuration was generated (Figure 55). In addition, a list of 52 linkers was generated by compiling linker sequences from multiple studies. The linker library was synthesized as an oligonucleotide pool, which was then expanded and randomly cloned into the entry vector via the Golden Gate assembly protocol, generating a plasmid library of LTRP5 variants covering a set of approximately 2700 possible linkers. Approximately 90 LTRP5 variant plasmids containing the linker sets were randomly selected and subjected to array screening, whereby each LTRP5 variant plasmid was co-transfected with a plasmid encoding a gRNA using scaffold 316 and a targeting spacer (see Table 58 for spacer sequences) into HEK293T cells seeded in a 96-well plate. Following transfection, cells were selected with puromycin and hygromycin for three days, and then cells were harvested and repression at the target locus was assessed by immunostaining of target cell surface markers followed by flow cytometry using an Attune ^™ NxT flow cytometer. For variant plasmids containing linker sets 1 to 28 (see Table 57 for sequences of linker combinations), LTRP inhibitory activity was evaluated for three different target loci ( B2M , Target 1, and Target 2). For linker sets 1 to 11, inhibition was measured 8, 15, and 45 days after transfection. For linker sets 12 to 28, inhibition was measured 7 and 17 days after transfection. For plasmids containing linker sets 31 to 89 and linker sets 1, 10, and 25 (Table 57), LTRP inhibitory activity was evaluated for the B2M locus 6, 13, and 24 days after transfection. Plasmids encoding LTRP5 molecules (without the ADD domain) and the original linker set (SEQ ID NO: 123 for L1; SEQ ID NO: 124 for L3A) were used as experimental controls. A non-targeting spacer was also included as a negative control. Table 57 : AA sequences of the approximately 90 linker sets evaluated in this example Connection sub-collection ID AA sequence of the first linker in the set ( position L3A) AA sequence of the second linker in the set ( position L1) SEQ ID NO. ( position L3A) SEQ ID NO. ( position L1) Collection 1 ASAAAPAAASAAASAPSAAAA ASAAAPAAA 3281 3286 Collection 36 PPTPSPSPVPSTPPTNSSSTPPTPS GSGNSSGSGGS 3280 3285 Collection 43 GSGGSGGSGGSPVPSTPPTPSPSTPPTP AFPAAPAPA 3279 3284 Collection 66 AASPAAPSAPPAAASP PPTP 3282 3287 Collection 85 ASPAAPAPASPAAPAPSAPAA IRAHGD 3278 3283 Collection 2 LQPVPPQALKREQVSQQ HIGGINS 22745 22748 Collection 3 GGGSGGGS GGGSGGGS 22746 22746 Collection 4 AASPAAPSAPPAAASP SPAGSPTSTEEGTSESAT 3282 22773 Collection 5 PGTSTEPSEGSAPGSP PPTP 22747 3287 Collection 6 HIGGINS GGGS 22748 22763 Collection 7 AASPAAPSAPPAAASP PPTPSPSPVP 3282 22768 Collection 8 ASPAAPSP ILGELT 22749 22774 Collection 9 GSGNSSGSGG HIGGINS 22750 22748 Collection 10 PPTPSPSPVPSTPPTNSSSTPPTPS SGSETPGTSESATPES 3280 22754 Collection 11 HIGGINS PPTPSPSPVP 22748 22768 Collection 12 EAAKEAAK PPTP 22751 3287 Collection 13 PPTP SWRVRYMVA 3287 22775 Collection 14 LRTRYEADLA GSGNSSGSGGS 22752 3285 Collection 15 EAAKEAAKEAAKEAAK PPTP 22753 3287 Collection 17 SGSETPGTSESATPES GGGSGGGS 22754 22746 Collection 19 GSGNSSGSGGS STEEGTSTEPSEGSAP 3285 22756 Collection 20 PPTPSPSPVSSTPPTPPTPS EAAKEAAK 22755 22751 Collection 21 STEEGTSTEPSEGSAP PPTPSPSPVP 22756 22768 Collection 23 GGGSGGGS YGAHLM 22746 22776 Collection 24 STEEGTSTEPSEGSAP GSGNSSGSGGSGGSGNSSGSGGS 22756 22777 Collection 25 PPTPSPSPVPSTPPTPPTPS GGGSGGGSGGGS 22757 22762 Collection 26 GGGSGGGSGGGSEAAKEAAKEAAK VQQKYKVSDTAATVTG 22758 22765 Collection 27 ASAAAPAAA GSGNSSGSGGSGGSGNSSGSGGS 3286 22777 Collection 28 PPTP GGGSGGGS 3287 22746 Collection 31 SGSETPGTSESATPES STEEGTSTEPSEGSAP 22754 22756 Collection 32 GSGGSGGSGGSPVPSTPPTPSPSTPPTP SPAGSPTSTEEGTSESAT 3279 22773 Collection 33 PPTP GGGS 3287 22763 Collection 34 ASPAAPAPA GGGS 22759 22763 Collection 37 GGGSGGGS GGGS 22746 22763 Collection 38 GGGSGGGS SGSETPGTSESATPES 22746 22754 Collection 39 GGGSGGGSEAAKEAAK STEEGTSTEPSEGSAP 22760 22756 Collection 40 PPTPSPSPVPSTPPTPPTPS GGGSGGGSGGGS 22757 22762 Collection 41 GGGSGGGSGGGSGGGS SPAGSPTSTEEGTSESAT 22761 22773 Collection 42 GSGNSSGSGGS AYVVSADEREGG 3285 22778 Collection 44 SGSETPGTSESATPES GSGNSSGSGGS 22754 3285 Collection 45 GGGSGGGSGGGS PGTSTEPSEGSAPGSP 22762 22747 Collection 46 GGGSGGGSGGGSGGGS EAAKEAAK 22761 22751 Collection 47 GSGNSSGSGGS PPTPSPSPVPSTPPTPPTPS 3285 22757 Collection 48 GGGSGGGSEAAKEAAK EAAKEAAKGGGSGGGS 22760 22771 Collection 49 GGGS GGGSGGGS 22763 22746 Collection 50 EAAK GSGGSGGSGGSPVPSTPPTPSPSTPPTP 22764 3279 Collection 51 EAAKEAAK EAAKEAAK 22751 22751 Collection 52 SGSETPGTSESATPES PPTP 22754 3287 Collection 53 GGGSGGGS PPTP 22746 3287 Collection 55 GGGSGGGSGGGS MIGP 22762 22779 Collection 56 VQQKYKVSDTAATVTG SGSETPGTSESATPES 22765 22754 Collection 57 GGGSGGGSGGVS GSGGSGGSGGSPVPSTPPTPSPSTPPTP 22766 3279 Collection 58 EAAKEAAKEAAKEAAK STEEGTSTEPSEGSAP 22753 22756 Collection 59 GGGSGGGS EAAKEAVKGGGSGGGS 22746 22780 Collection 60 GGGSGGGSGGGSGGGS PPTP 22761 3287 Collection 61 EAAK EAAKEAAKGGGSGGGS 22764 22771 Collection 62 GGGS PPTPSPSPVP 22763 22768 Collection 63 EAAKEAVKEAAKEAAK HIGGINS 22767 22748 Collection 64 PPTPSPSPVPSTPPTPPTPS GGGSGGGS 22757 22746 Collection 65 PPTPSPSPVP PPTP 22768 3287 Collection 67 STEEGTSTEPSEGSAP PPTP 22756 3287 Collection 68 GGGSGGGSGGGS PPTPSPSPVPSTPPTNSSSTPPTPS 22762 3280 Collection 69 PPTPSPSPVP PGTSTEPSEGSAPGSP 22768 22747 Collection 70 ASPAAPAPA LMPYEL 22759 22781 Collection 71 PPTPSPSPVP PPTP 22768 3287 Collection 72 GGGSGGGSGGGS ASAAAPAAA 22762 3286 Collection 73 GGGSGGGS STEEGTSTEPSEGSAP 22746 22756 Collection 74 PPTPSPSPVP HIGGINS 22768 22748 Collection 75 EAAKEAAK HIGGINS 22751 22748 Collection 76 PPTPSPSPVPSTPPTPPTPS SPTP 22757 22782 Collection 77 EAAKEAAKEAAKEAAK GGGSGGGSGGGSGGGSGGGS 22753 22742 Collection 78 EAAKEAAK PPTPSPSPVPSTPPTPPTPS 22751 22757 Collection 79 PPTPSPSPVP GGGSGGGSGGGSGGGS 22768 22761 Collection 80 ITHKASSRMTFA GGGSGGGSGGGS 22769 22762 Collection 81 STEEGTSTEPSEGSAP GCGS 22756 22783 Collection 82 ASSAAPAPA GGGS 22770 22763 Collection 83 STEEGTSTEPSEGSAP GGGSGGGS 22756 22746 Collection 84 SGSETPGTSESATPES GGGSGGGSGGGSGGGSGGGS 22754 22742 Collection 86 EAAKEAAKGGGSGGGS EAAKEAAKGGGSGGGS 22771 22771 Collection 87 EAAKEAAKEAAKEAAK NKQNIWQA 22753 22784 Collection 88 GGGSGGGSGGGS ASAAAPAAA 22762 3286 Collection 89 AASPAAPSAPPASASP PPMPSPSPVP 22772 22785 Table 58 : Sequences of spacers targeting the B2M locus Spacer ID Target Targeting spacer sequence (RNA) SEQ ID NO 7.165 B2M UCCCUAUGUCCUUGCUGUUU 3114 result:

An array screen was performed to evaluate the level of repression activity at endogenous target loci in HEK293T cells transfected with LTRP5 variant plasmids with different linker combinations. LTRP5 plasmids containing linker sets 1 to 11 were measured for repression at B2M , Target 1, and Target 2 loci at 8, 15, and 45 days post-transfection, and the results are shown in Figures 56 to 58. The data indicate that the best performing linker combination varies with the specific target. Of the 11 linker combinations analyzed in this experiment, the use of linker sets 1 and 10 consistently induced the highest levels of repression at both B2M and Target 2 loci overall, with linker set 10 showing the second highest linker set tested at the Target 1 locus. Subsequently, linker 1 was used as a benchmark in the next two experiments analyzing linker sets 12 to 28 and linker sets 31 to 89.

LTRP5 plasmids containing linker sets 12 to 28 were measured for repression at the B2M , Target 1, and Target 2 loci 7 and 17 days after transfection, with linker set 1 from the previous experiment serving as the benchmark, and the results are depicted in Figures 59 to 60. The data showed that, similar to what was observed in the previous experiment, the best performing linker set varied by target. Of the 17 linker combinations analyzed in this second experiment, the use of linker sets 1 and 25 elicited the highest levels of repression at two of the three target loci ( B2M and Target 1 for set 1; B2M and Target 2 for set 25).

LTRP5 plasmids containing linker sets 31 to 89 were measured for repression at the B2M locus 6, 13, and 24 days after transfection, with linker sets 1, 10, and 25 from the first two experiments serving as a benchmark, and the results are depicted in Tables 59 to 61. The data showed that at 24 days after transfection, the use of linker sets 85, 43, 36, 10, 64, 1, 40, 25, 66, 76, and 52 resulted in at least a 50% increase in repressive activity relative to the activity level of the LTRP5 control when paired with the B2M spacer (Tables 59 to 61). Next, five of these 11 best performing linker sets were selected for future validation based on the original linker set sequences (SEQ ID NO: 123 for L1; SEQ ID NO: 124 for L3A) that were different from the other best candidate combinations and linkers. Table 59 : Levels of B2M inhibition mediated by LTRP5 constructs with linker sets 1 , 10 , 25 , and 31-39 quantified 6 days after transfection Structure Connect sub-collection Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5 Original/Baseline NT 4.607 0.289 3 LTRP5 Collection 54 7.165 31.000 2.955 3 LTRP5 Collection 69 7.165 33.400 1.015 3 LTRP5 Collection 42 7.165 35.167 1.258 3 LTRP5 Collection 89 7.165 34.733 0.603 3 LTRP5 Original/Baseline 7.165 38.667 0.379 3 LTRP5 Collection 61 7.165 48.300 1.931 3 LTRP5 Collection 59 7.165 49.467 0.709 3 LTRP5 Collection 80 7.165 48.633 0.643 3 LTRP5 Collection 33 7.165 53.800 1.200 3 LTRP5 Collection 37 7.165 55.167 0.416 3 LTRP5 Collection 51 7.165 51.900 0.819 3 LTRP5 Collection 49 7.165 52.267 0.723 3 LTRP5 Collection 48 7.165 50.500 1.082 3 LTRP5 Collection 62 7.165 53.367 0.493 3 LTRP5 Collection 41 7.165 53.067 0.473 3 LTRP5 Collection 50 7.165 53.400 0.458 3 LTRP5 Collection 38 7.165 53.833 0.231 3 LTRP5 Collection 78 7.165 52.633 0.379 3 LTRP5 Collection 86 7.165 53.833 1.484 3 LTRP5 Collection 53 7.165 55.167 0.764 3 LTRP5 Collection 87 7.165 55.533 0.833 3 LTRP5 Collection 46 7.165 53.033 0.153 3 LTRP5 Collection 55 7.165 50.633 1.650 3 LTRP5 Collection 34 7.165 57.800 0.794 3 LTRP5 Collection 75 7.165 53.400 0.794 3 LTRP5 Collection 39 7.165 55.467 1.002 3 LTRP5 Collection 56 7.165 54.000 1.114 3 LTRP5 Collection 60 7.165 57.933 1.328 3 LTRP5 Collection 82 7.165 57.500 0.889 3 LTRP5 Collection 73 7.165 53.433 0.907 3 LTRP5 Collection 83 7.165 59.333 1.069 3 LTRP5 Collection 63 7.165 55.400 0.872 3 LTRP5 Collection 88 7.165 58.567 1.436 3 LTRP5 Collection 68 7.165 55.633 0.231 3 LTRP5 Collection 58 7.165 54.433 0.723 3 LTRP5 Collection 79 7.165 56.700 0.520 3 LTRP5 Collection 70 7.165 55.467 1.350 3 LTRP5 Collection 32 7.165 58.467 0.723 3 LTRP5 Collection 77 7.165 56.133 0.651 3 LTRP5 Collection 81 7.165 60.400 0.500 3 LTRP5 Collection 57 7.165 57.800 1.153 3 LTRP5 Collection 44 7.165 57.933 1.617 3 LTRP5 Collection 47 7.165 55.400 0.173 3 LTRP5 Collection 31 7.165 59.767 0.751 3 LTRP5 Collection 74 7.165 58.067 0.569 3 LTRP5 Collection 65 7.165 60.300 0.954 3 LTRP5 Collection 84 7.165 59.333 0.902 3 LTRP5 Collection 67 7.165 60.767 1.779 3 LTRP5 Collection 72 7.165 57.433 0.723 3 LTRP5 Collection 71 7.165 59.467 0.379 3 LTRP5 Collection 52 7.165 61.533 0.153 3 LTRP5 Collection 76 7.165 62.900 0.400 3 LTRP5 Collection 66 7.165 61.933 0.379 3 LTRP5 Collection 25 7.165 61.033 0.416 3 LTRP5 Collection 40 7.165 62.800 0.889 3 LTRP5 Collection 1 7.165 58.133 0.058 3 LTRP5 Collection 64 7.165 57.400 1.229 3 LTRP5 Collection 10 7.165 59.967 1.222 3 LTRP5 Collection 36 7.165 65.333 0.902 3 LTRP5 Collection 43 7.165 64.800 1.253 3 LTRP5 Collection 85 7.165 64.333 1.274 3 Table 60 : B2M inhibition levels mediated by LTRP5 constructs with linker sets 1 , 10 , 25 , and 31 to 39 quantified 13 days after transfection Structure Connect sub-collection Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5 Original/Baseline NT 1.877 0.315 3 LTRP5 Collection 54 7.165 2.670 0.866 3 LTRP5 Collection 69 7.165 1.200 0.190 3 LTRP5 Collection 42 7.165 24.000 0.794 3 LTRP5 Collection 89 7.165 25.500 0.755 3 LTRP5 Original/Baseline 7.165 30.800 0.917 3 LTRP5 Collection 61 7.165 31.600 0.700 3 LTRP5 Collection 59 7.165 32.267 0.473 3 LTRP5 Collection 80 7.165 32.767 0.231 3 LTRP5 Collection 33 7.165 36.833 1.137 3 LTRP5 Collection 37 7.165 35.900 1.179 3 LTRP5 Collection 51 7.165 34.767 0.723 3 LTRP5 Collection 49 7.165 36.167 0.929 3 LTRP5 Collection 48 7.165 36.267 1.274 3 LTRP5 Collection 62 7.165 34.433 0.551 3 LTRP5 Collection 41 7.165 35.067 1.484 3 LTRP5 Collection 50 7.165 38.067 0.874 3 LTRP5 Collection 38 7.165 36.567 0.351 3 LTRP5 Collection 78 7.165 35.000 0.700 3 LTRP5 Collection 86 7.165 34.933 1.747 3 LTRP5 Collection 53 7.165 35.833 1.234 3 LTRP5 Collection 87 7.165 35.800 1.015 3 LTRP5 Collection 46 7.165 36.200 0.361 3 LTRP5 Collection 55 7.165 36.033 1.950 3 LTRP5 Collection 34 7.165 40.733 1.168 3 LTRP5 Collection 75 7.165 36.033 1.604 3 LTRP5 Collection 39 7.165 38.867 1.266 3 LTRP5 Collection 56 7.165 37.467 0.764 3 LTRP5 Collection 60 7.165 40.467 1.750 3 LTRP5 Collection 82 7.165 38.567 1.069 3 LTRP5 Collection 73 7.165 35.433 1.845 3 LTRP5 Collection 83 7.165 39.100 0.889 3 LTRP5 Collection 63 7.165 37.433 1.002 3 LTRP5 Collection 88 7.165 39.033 1.930 3 LTRP5 Collection 68 7.165 37.833 0.493 3 LTRP5 Collection 58 7.165 38.700 0.866 3 LTRP5 Collection 79 7.165 39.367 0.153 3 LTRP5 Collection 70 7.165 37.467 1.701 3 LTRP5 Collection 32 7.165 42.833 0.289 3 LTRP5 Collection 77 7.165 38.767 0.907 3 LTRP5 Collection 81 7.165 39.300 1.039 3 LTRP5 Collection 57 7.165 41.233 1.779 3 LTRP5 Collection 44 7.165 40.667 0.814 3 LTRP5 Collection 47 7.165 39.667 0.850 3 LTRP5 Collection 31 7.165 43.433 1.650 3 LTRP5 Collection 74 7.165 40.467 0.802 3 LTRP5 Collection 65 7.165 42.000 0.608 3 LTRP5 Collection 84 7.165 41.700 0.200 3 LTRP5 Collection 67 7.165 41.600 1.277 3 LTRP5 Collection 72 7.165 39.400 0.781 3 LTRP5 Collection 71 7.165 42.533 0.153 3 LTRP5 Collection 52 7.165 44.333 0.635 3 LTRP5 Collection 76 7.165 45.033 1.721 3 LTRP5 Collection 66 7.165 43.233 0.833 3 LTRP5 Collection 25 7.165 49.233 0.231 3 LTRP5 Collection 40 7.165 47.767 1.002 3 LTRP5 Collection 1 7.165 48.100 0.917 3 LTRP5 Collection 64 7.165 44.133 1.097 3 LTRP5 Collection 10 7.165 48.733 1.069 3 LTRP5 Collection 36 7.165 48.833 1.358 3 LTRP5 Collection 43 7.165 49.267 1.222 3 LTRP5 Collection 85 7.165 46.933 2.312 3 Table 61 : B2M inhibition levels mediated by LTRP5 constructs with linker sets 1 , 10 , 25 , and 31 to 39 quantified 24 days after transfection Structure Connect sub-collection Spacer Average HLA- negative cells% Standard Deviation Sample size LTRP5 Original/Baseline NT 1.290 0.098 3 LTRP5 Collection 54 7.165 3.670 1.105 3 LTRP5 Collection 69 7.165 4.270 0.282 3 LTRP5 Collection 42 7.165 22.600 1.153 3 LTRP5 Collection 89 7.165 24.500 0.781 3 LTRP5 Original/Baseline 7.165 25.067 0.907 3 LTRP5 Collection 61 7.165 27.100 0.781 3 LTRP5 Collection 59 7.165 27.167 0.351 3 LTRP5 Collection 80 7.165 28.367 1.185 3 LTRP5 Collection 33 7.165 28.367 0.208 3 LTRP5 Collection 37 7.165 28.833 0.751 3 LTRP5 Collection 51 7.165 28.933 0.551 3 LTRP5 Collection 49 7.165 28.967 1.950 3 LTRP5 Collection 48 7.165 29.467 0.757 3 LTRP5 Collection 62 7.165 29.633 0.551 3 LTRP5 Collection 41 7.165 29.767 1.457 3 LTRP5 Collection 50 7.165 30.000 0.436 3 LTRP5 Collection 38 7.165 30.167 1.050 3 LTRP5 Collection 78 7.165 30.200 1.480 3 LTRP5 Collection 86 7.165 30.367 1.041 3 LTRP5 Collection 53 7.165 30.567 2.055 3 LTRP5 Collection 87 7.165 30.733 1.258 3 LTRP5 Collection 46 7.165 31.033 0.723 3 LTRP5 Collection 55 7.165 31.167 2.413 3 LTRP5 Collection 34 7.165 31.833 0.802 3 LTRP5 Collection 75 7.165 31.867 2.892 3 LTRP5 Collection 39 7.165 31.867 0.874 3 LTRP5 Collection 56 7.165 32.267 0.681 3 LTRP5 Collection 60 7.165 32.733 1.206 3 LTRP5 Collection 82 7.165 32.967 1.274 3 LTRP5 Collection 73 7.165 33.133 0.351 3 LTRP5 Collection 83 7.165 33.333 0.569 3 LTRP5 Collection 63 7.165 33.400 2.551 3 LTRP5 Collection 88 7.165 33.500 1.803 3 LTRP5 Collection 68 7.165 33.533 0.666 3 LTRP5 Collection 58 7.165 33.633 1.266 3 LTRP5 Collection 79 7.165 34.000 0.100 3 LTRP5 Collection 70 7.165 34.167 2.836 3 LTRP5 Collection 32 7.165 34.167 1.193 3 LTRP5 Collection 77 7.165 34.467 0.814 3 LTRP5 Collection 81 7.165 34.733 0.404 3 LTRP5 Collection 57 7.165 34.833 2.250 3 LTRP5 Collection 44 7.165 34.933 1.012 3 LTRP5 Collection 47 7.165 35.167 0.802 3 LTRP5 Collection 31 7.165 35.433 2.060 3 LTRP5 Collection 74 7.165 35.833 0.651 3 LTRP5 Collection 65 7.165 36.467 0.681 3 LTRP5 Collection 84 7.165 37.167 1.069 3 LTRP5 Collection 67 7.165 37.300 1.311 3 LTRP5 Collection 72 7.165 37.667 0.850 3 LTRP5 Collection 71 7.165 37.733 0.681 3 LTRP5 Collection 52 7.165 38.767 0.929 3 LTRP5 Collection 76 7.165 39.167 1.858 3 LTRP5 Collection 66 7.165 39.367 0.503 3 LTRP5 Collection 25 7.165 40.400 1.808 3 LTRP5 Collection 40 7.165 40.633 1.629 3 LTRP5 Collection 1 7.165 41.000 1.127 3 LTRP5 Collection 64 7.165 41.367 1.877 3 LTRP5 Collection 10 7.165 42.167 2.113 3 LTRP5 Collection 36 7.165 42.333 1.250 3 LTRP5 Collection 43 7.165 42.433 0.666 3 LTRP5 Collection 85 7.165 42.500 2.272 3

The experiments show that the inhibitory activity of LTRP molecules can be further enhanced by changing the linker sequence located between key domains of the LTRP molecule. Here, optimizing the linker sequence at two locations, namely between the DNMT3L interaction domain and the inhibitory subdomain and between the inhibitory subdomain and the catalytically inactive CasX, resulted in substantial improvements in the inhibitory activity of the LTRP molecule. Example 14 : Evaluation of the use of the ADD domain in LTRP in a mouse model

Experiments were performed to assess the persistence of methylation following treatment with the CasX:gRNA system when delivered in vivo in a mouse model. Methylation persistence of LTRP1-targeted molecules, mPCSK9 transcript attenuation, and mPCSK9 protein production were assessed in the presence and absence of the ADD domain. Materials and Methods: LNP Preparation:

LTRP mRNA and gRNA were encapsulated into LNPs using a custom T-mixer micromixing device with a flow rate of 20 mL/min and a 3:1 aqueous to organic phase mixing ratio using an ALC-0315-based lipid mixture. The composition was as follows: 50:10:38.5:1.5 mol% ionizable lipid:DSPC:cholesterol:DMG-PEG2000. Briefly, to formulate LNPs, equal mass ratios of LTRP mRNA and gRNA were diluted in 25 mM sodium acetate (pH 4.0). A 10 mM concentration of lipid mixture was prepared in anhydrous ethanol. mRNA/gRNA co-formulations were generated using a predetermined N/P ratio. Using a syringe pump infusion device, RNA and lipids were passed through a custom T-mixer device at a predetermined flow rate ratio. After formulation, LNPs were dialyzed into PBS (pH 7.4) to reduce ethanol concentration and increase pH, thereby improving particle stability. Buffer exchange of mRNA/gRNA-LNPs was achieved by dialysis into PBS (pH 7.4) overnight at 4°C using a 10k Slide-A-Lyzer™ Dialysis Cassette (Thermo Scientific™) or 12-14 kDa Dialysis Tubes (Repligen). After dialysis, mRNA/gRNA-LNPs were concentrated to >0.2 mg/mL using a 30-100 kDa Amicon®-Ultra centrifugal filter (Millipore) and subsequently sterile filtered using an Acrodisc PES membrane filter. The formulated LNPs were analyzed on a Malvern Zetasizer to determine their diameter and polydispersity index (PDI). Encapsulation efficiency and RNA concentration were determined by RiboGreen™ analysis using Invitrogen's Quant-iT™ RiboGreen™ RNA Assay Kit. The above LNPs were used in various experiments to deliver LTRP mRNA and gRNA to target tissues in vivo. LNPs for in vivo delivery of encapsulated CasX mRNA and targeting gRNA:

To assess the effects of using LTRP1A and LTRP1B in vivo, LTRP mRNA (see Table 62) and gRNA targeting mPCSK9 using scaffold 316 v1 and spacer 27.94 (SEQ ID: 3330, Table 63) were encapsulated in the same LNP using a 1:1 mRNA:gRNA mass ratio. The formulated LNPs were exchanged with PBS for intravenous injection. Briefly, 3.0 mg/kg of LNPs were administered intravenously to 4-week-old C57BL/6 mice via the tail vein. Mice were observed for five minutes after injection to ensure recovery from anesthesia and then placed in cages. Animals injected with vehicle (PBS) served as negative experimental controls and mice injected with mRNA:gRNA encoding LTRP1 and above targeting mPCSK9 served as positive controls. Baseline was obtained by fasting blood draw. Blood was drawn every three weeks. Seven, fourteen and forty-two days after administration, three mice under each condition were euthanized and blood and liver tissue were collected. Serum was collected for mPCSK9 ELISA, and liver tissue was homogenized for mRNA extraction and gDNA extraction using the Zymo Research Quick DNA/RNA Miniprep kit according to the manufacturer's instructions. Enzymatic methylation sequencing (EM-seq):

To determine the level of on-target methylation at the PCSK9 locus, gDNA was extracted from the collected tissue using the Zymo Quick-DNA Miniprep Plus kit according to the manufacturer's instructions. The extracted gDNA was then subjected to enzymatic methylation conversion using the Enzymatic Methyl-Seq Conversion Module (NEB) to convert any unmethylated cytosine to uracil according to the manufacturer's protocol. The resulting processed DNA was then sequenced using next-generation sequencing (NGS) to determine the level of on-target methylation.

NGS processing and analysis:

The target amplicon was amplified by PCR from 50 ng of EM-treated DNA using a set of primers specific for the target location of interest for EM conversion (mouse PCSK9 locus). These gene-specific primers contain additional sequence at the 5′ end to introduce the Illumina ^™ adapter. The amplified DNA product was purified using the Cytiva Sera-Mag Select DNA Cleanup Kit. The quality and quantification of the amplicon were assessed using the Fragment Analyzer DNA Analysis Kit (Agilent, dsDNA 35 to 1500 bp). The amplicon was sequenced on an Illumina ^™ Miseq ^™ according to the manufacturer's instructions. The sequenced raw fastq files were processed using the Bismark bisulfite read mapping program and methylation call program. PCR amplification of EM-treated DNA will convert all uracil nucleotides to thymine, and sequencing of the PCR products will determine the rate of cytosine to thymine conversion, which is a readout of the level of target methylation at the PCSK9 locus mediated by each LTRP molecule. qPCR analysis of mPCSK9 mRNA attenuation:

After necropsy, liver pieces were rapidly frozen. These liver pieces were then immersed in an excess volume of Zymo DNA/RNA Shield reagent and homogenized using a bullet blender (a device with ceramic or steel beads in the tube). After homogenization, the lysate was treated with proteinase K. mRNA from proteinase K-treated liver lysate was extracted using the Zymo Quick RNA Miniprep Kit (Catalog No. R1055) according to the manufacturer's instructions and analyzed using Taqman Gene Expression Assays using mPCSK9 and eukaryotic 18S probes according to the manufacturer's instructions. ELISA Method:

Serum from animal samples was analyzed using the Legend Max Mouse PCSK9 ELISA Kit (BioLegend Catalog No. 443207) according to the manufacturer's instructions. Results:

To assess the persistence of methylation following treatment with the CasX:gRNA system when delivered in vivo in a mouse model, mRNA encoding LTRP1 (SEQ ID NO. 3333), LTRP1A (SEQ ID NO: 6528), or LTRP1B (SEQ ID NO: 9741) and gRNA 316v1.27.94 (SEQ ID NO: 3330) were encapsulated in LNPs and injected into C57BL/6 mice. Quantification of the resulting PCSK9 mRNA attenuation is shown in Table 64. The data showed that at 7 and 14 days, all three LTRP1 targeting molecules had similar levels of attenuation, with LTRP1 and LTRP1A showing higher levels of inhibition at day 42. Methylation data from EM-seq (Figure 62) showed high methylation levels for all three LTRP1-directed molecules tested, supporting this result, with a slight decrease in the total methylation percentage at days 14 and 42 in mice treated with LTRP1 and LTRP1B.

Quantification of PCSK9 secretion showed that all LTRP molecules tested had similar levels of PCSK9 inhibition at day 7 (week 1, Table 65). Compared to LTRP1, LTRP1A showed increased persistence of inhibition at week 6 of testing, while LTRP1B showed increased persistence of inhibition at week 8.

These experiments demonstrate that LTRP1 targeting molecules delivered in mRNA form via LNPs in the presence and absence of the ADD domain are able to induce efficient silencing in vivo. Table 62 : mRNA sequences of LTRP constructs tested in this example LTRP SEQ ID NO. Components LTRP1 3333 LTRP1 ZIM3, 80 polyadenylation, SV40, C-terminal 2xFLAG LTRP1A 6528 LTRP1 ZIM3, split polyadenylation, SV40 LTRP1B 9741 LTRP1 ZIM3, ADD, split polyadenylation, SV40 Table 63 : gRNA and targeting sequences used in this example gRNA ID SEQ ID NO. 27.94 3071 316v1.27.94 3330 Table 64 : Liver lysate mRNA qPCR Days after medication LTRP 7 days 14 days 42 days average value std average value std average value std Medium (N=3) 7.14 49.81 5.24 42.63 1.00 17.10 LTRP1 (N=3) -96.55 1.77 -96.96 1.21 -93.65 2.60 LTRP1A (N=3) -99.04 0.66 -99.76 0.09 -93.19 4.73 LTRP1B (N=3) -96.13 1.47 -92.39 5.65 -80.55 5.79 Table 65 : Changes in secreted PCSK9 measured by ELISA Percent PCSK9 secretion determined by serum ELISA normalized to baseline Research Week Medium LTRP1 LTRP1A LTRP1B average value SEM average value SEM average value SEM average value SEM 1 23.80 45.23 -94.45 0.45 -93.50 0.91 -94.30 0.60 2 -15.06 14.48 -92.20 1.96 -85.10 7.07 -93.69 0.44 5 4.98 10.38 -90.18 2.78 -95.80 0.78 -93.37 0.42 6 -11.92 27.13 -92.65 1.72 -95.81 0.79 -90.36 2.14 8 17.84 9.53 -80.96 6.72 NA NA -90.58 1.20 The values below the LOQ of the assay were set as the LOQ (6.25 ng/mL) and the LTRP1A week 8 data were not collected. Example 15 : Efficacy and persistence of LTRPs with RD1 domains

Experiments were performed to assess the persistence of methylation in a mouse model after in vivo treatment with LTRP1-targeted molecules containing the RD1 domain. Materials and Methods:

LNPs were prepared as described in Example 14.

In vivo experiments examined the efficacy of LTRP1 targeting molecules containing the King Cobra RD1 domain (SEQ ID NO. 3339). LTRP1A-Cobra and LTRP1B-Cobra (SEQ ID NO. 6530, SEQ ID NO. 9743) were constructed by replacing the RD1 domain with the ZIM3 domain in LTRP1A and LTRP 1B. LNPs containing LTRP mRNA (Table 66) and gRNA targeting mPCSK9 (SEQ ID: 3330, Table 66) were formulated and exchanged with PBS for intravital injection. Briefly, LNPs were administered intravenously to 4-week-old C57BL/6 mice via the tail vein. Mice were observed for five minutes after injection to ensure recovery from anesthesia and then placed in cages. Animals injected with vehicle (PBS) served as negative experimental controls and mice injected with mRNA:gRNA encoding LTRP1A (SEQ ID: 6528) and gRNA targeting mPCSK9 using scaffold 316 and spacer 27.94 (SEQ ID: 3330) served as positive controls. Baseline was obtained by fasting blood draw. Blood was drawn once every three weeks. Seven and forty-two days after administration, mice were euthanized and blood and liver tissue were collected. Serum was collected for mPCSK9 ELISA, and liver tissue was homogenized for mRNA extraction and gDNA extraction using the Zymo Research Quick DNA/RNA Miniprep Kit according to the manufacturer's instructions. Enzymatic methylation sequencing (EM-seq):

To determine the level of on-target methylation at the PCSK9 locus, gDNA was extracted from the collected tissue using the Zymo Quick-DNA Miniprep Plus Kit according to the manufacturer's instructions. The extracted gDNA was then subjected to enzymatic methylation conversion using the Enzymatic Methyl-Seq Conversion Module (NEB) to convert any unmethylated cytosine to uracil according to the manufacturer's protocol. The resulting processed DNA was then sequenced using next-generation sequencing (NGS) to determine the level of on-target methylation. NGS Processing and Analysis:

The target amplicon was amplified by PCR from 50 ng of EM-treated DNA using a set of primers specific for the target location of interest for EM conversion (mouse PCSK9 locus). These gene-specific primers contain additional sequence at the 5′ end to introduce the Illumina ^™ adapter. The amplified DNA product was purified using the Cytiva Sera-Mag Select DNA Cleanup Kit. The quality and quantification of the amplicon were assessed using the Fragment Analyzer DNA Analysis Kit (Agilent, dsDNA 35 to 1500 bp). The amplicon was sequenced on an Illumina ^™ Miseq ^™ according to the manufacturer's instructions. The sequenced raw fastq files were processed using the Bismark bisulfite read mapping program and methylation call program. PCR amplification of EM-treated DNA will convert all uracil nucleotides to thymine, and sequencing of the PCR products will determine the rate of cytosine to thymine conversion, which is a readout of the level of target methylation at the PCSK9 locus mediated by each LTRP molecule. qPCR analysis of mPCSK9 mRNA attenuation:

After necropsy, liver pieces were rapidly frozen. These liver pieces were then immersed in an excess volume of Zymo DNA/RNA Shield reagent and homogenized using a bead blender (Bullet Blender instrument with ceramic or steel beads in the tube). After homogenization, the lysate was treated with proteinase K. mRNA from proteinase K-treated liver lysate was extracted using the Zymo Quick RNA Miniprep Kit (Catalog No. R1055) according to the manufacturer's instructions and analyzed using TaqMan Gene Expression Assays with mPCSK9 and eukaryotic 18S probes according to the manufacturer's instructions. ELISA Method:

Serum from animal samples was analyzed using the LEGEND MAX Mouse PCSK9 ELISA Kit (BioLegend Catalog No. 443207) according to the manufacturer's instructions. Results:

TSS-proximal DNA methylation levels measured by expander enzymatic methylation sequencing (EM-seq) from gDNA extracted from homogenized livers of N=3 mice sacrificed on day 7 post-treatment showed similar methylation levels for all constructs, with higher doses tending to have higher median methylation (Figure 63).

Table 67 Quantitative serum PCSK9 ELISA results, which show that all LTRP1 constructs showed similar initial effects, and the groups given 0.75 mpk or 1.5 mpk had serum PCSK9 attenuated by about 90% 42 days after dosing.

Table 68 Results of quantitative mPCSK9 mRNA qPCR analysis showing that all three LTRP1 constructs maintained robust transcript reductions of more than 80% attenuation at both doses. LTRP1A, LTRP1A-cobra, and LTRP1B-cobra exhibited potent and sustained effects on mRNA transcript levels until 42 days post-dose. At 42 days, LTRP1A and LTRP1A-cobra showed a slight increase in mPCSK9 expression at a lower dose (0.75 mg/kg), but it is unclear whether this represents a trend.

These experiments demonstrate that LTRP1-targeted molecules can produce persistent methylation, leading to reduced expression of PCSK9 with the RD1 domain in a mouse model. Table 66 : Sequences of mRNAs and gRNAs evaluated mRNA SEQ ID NO. gRNA Dosage mg/kg LTRP1A 6528 316v1 27.94 (SEQ ID NO. 3330) 1.5 0.75 LTRP1A-Cobra 6530 1.5 0.75 LTRP1B-Cobra 9743 1.5 0.75 PBS (negative control) NA NA NA Table 67 : Serum PCSK9 ELISA performed on Day 7 , Day 14 , Day 35 and Day 42 * Change in serum PCSK9 relative to baseline % Vehicle (PBS) Vehicle (PBS) sky average value SEM n average value SEM n 0 0 0 9 N/A N/A N/A 7 50.87 79.48 3 N/A N/A N/A 14 89.78 86.04 6 N/A N/A N/A 35 -19.45 80.54 6 N/A N/A N/A 42 111.26 6.19 2 N/A N/A N/A LTRP1A- Cobra (0.75 mpk) LTRP1A- Cobra (1.5 mpk) sky average value SEM n average value SEM n 0 0 0 9 0 0 9 7 -90.42 3.47 3 -87.40 2.37 3 14 -87.51 9.17 6 -87.03 5.74 6 35 -83.73 12.37 6 -87.03 5.74 6 42 -91.31 2.87 2 -93.43 1.15 2 LTRP1B- Cobra (0.75 mpk) LTRP1B- Cobra (1.5 mpk) sky average value SEM n average value SEM n 0 0 0 9 0 0 9 7 -90.22 4.27 3 -87.99 4.45 3 14 -88.43 6.86 6 -91.63 2.61 6 35 -89.11 3.79 6 -91.63 2.61 6 42 -88.33 1.86 2 -90.18 2.72 2 LTRP1A (0.75 mpk) LTRP1A (1.5 mpk) sky average value SEM n average value SEM n 0 0 0 9 0 0 9 7 -91.57 1.12 3 -87.94 2.11 3 14 -88.21 4.21 6 -90.87 2.15 6 35 -86.95 4.41 6 -90.87 2.15 6 42 -70.89 21.14 2 -89.01 1.36 2 * Data for day 7 are from animals sampled at that time point only. Data for days 7 and 35 are from different individuals that survived until day 42. Data for day 42 are from animals sampled at that time point only. Table 68 : Percent inhibition of mPCSK9 mRNA at days 7 and 42 as determined by qPCR Percentage of mPCSK9 transcript/18S inhibition Medium Medium sky average value SEM n average value SEM n 0 0 0 3 N/A N/A N/A 7 19.65 49.23 3 N/A N/A N/A 42 9.38 44.25 2 N/A N/A N/A LTRP1A- Cobra (0.75 mpk) LTRP1A- Cobra (1.5 mpk) sky average value SEM n average value SEM n 0 0 0 3 0 0 3 7 -92.90 1.48 3 -96.68 2.01 3 42 -79.37 0.57 2 -87.63 6.35 2 LTRP1B- Cobra (0.75 mpk) LTRP1B- Cobra (1.5 mpk) sky average value SEM n average value SEM n 0 0 0 3 0 0 3 7 -91.54 4.95 3 -98.15 0.55 3 42 -90.86 0.57 2 -96.02 1.31 2 LTRP1A (0.75 mpk) LTRP1A (1.5 mpk) sky average value SEM n average value SEM n 0 0 0 3 0 0 3 7 -92.79 1.21 3 -95.36 1.81 3 42 -84.55 5.15 2 -91.95 4.47 2 Example 16 : Functional evaluation of the CasX:gRNA system targeting the human PCSK9 locus

Experiments were performed to demonstrate that LNPs delivering encapsulated LTRP6 mRNA and PCSK9 targeting gRNA induce repression at the endogenous human PCSK9 locus in primary human hepatocytes (PHH) and thus induce a decrease in secreted PCSK9 and PCSK9 mRNA. Four LTRP molecules (Table 69) and the guiding scaffold 316v1 (EQ ID NO: 2968) were selected for evaluation in this example. Materials and Methods:

PCSK9 Attenuation in Primary Human Hepatocytes The first four edited targeting sequences (Table 69) were transfected into PHH cells by liposomal transfection to assess PCSK9 secretion and mRNA reduction. Dose response curves were generated from two batches of PHH cells, with each condition run for three rounds in triplicate wells. The highest tested dose of 2400 ng/well of total encapsulated RNA was used to measure changes in mRNA expression between treated and naive cells by RNA-seq. Synthesis of gRNA:

gRNA targeting the human PCSK9 locus was chemically synthesized as a chemically modified v1 version for delivery. The sequences targeting PCSK9 are listed in Table 69.

Production of LTRP mRNA by IVT. Briefly, constructs encoding the 5'UTR region, the codon-optimized LTRP, and the 3'UTR region were cloned into plasmids containing the T7 promoter and a polyadenylation tail. The resulting plasmids were linearized and subsequently used in IVT reactions using CleanCap® AG and N1-methyl-pseudouridine. Formulation of lipid nanoparticles (LNPs):

LTRP mRNA and targeting gRNA were encapsulated in LNPs made from an ionizable lipid mixture containing ALC0315 ionizable lipid:18:0 PC DSPC):cholesterol:DMG-PEG2000 at N/P 6 using an in-house custom-made T-mixer. Briefly, to formulate LNPs, split formulations (containing mRNA or sgRNA only) were diluted at a fixed ratio for co-formulation or separately for split formulations in 25 mM sodium acetate buffer, pH 4.0. ALC315 ionizable lipids were mixed with the above lipids at a molar ratio of 50:10:38.5:1.5% using absolute ethanol. The RNA and lipid phases were passed through a custom-made T-type mixer at a flow rate ratio of 3:1 and a flow rate of 20 ml/min. After formulation, the LNPs were transferred and dialyzed using a 10 KDa membrane dialysis cartridge (Thermo Scientific ^™ ) and buffer exchanged into 1× PBS to reduce the ethanol concentration and increase the pH to 7.4, thereby forming mature and stable particles. After dialysis, the RNA-LNPs were buffer exchanged into PBS (pH 7.4) storage buffer containing 300 mM sucrose and concentrated to the appropriate concentration using a 100 kDa Amicon®-Ultra centrifugal filter (Millipore) and sterile filtered. The formulated LNPs were then subjected to one freeze-thaw cycle at -80°C and analyzed on a Stunner (Unchained Labs) to determine their mean particle size (d. nm.) and polydispersity index (PDI). Encapsulation efficiency and RNA concentration were determined by RiboGreen™ analysis using Invitrogen's Quant-iT™ RiboGreen™ RNA Assay Kit. In each of the experiments described herein, LNPs were used to deliver mRNA and gRNA to target cells and tissues by mixing a 1:1 mass ratio of LNPs containing mRNA and LNPs containing gRNA. Delivery of LNPs encapsulating CasX mRNA and targeting gRNA into primary human hepatocytes:

Two batches (lot 271 and lot 31) of primary human hepatocytes (Lonza Biologics) were plated at a density of 65K cells/well. Twenty-four hours later, each plate was stamped with a single formulation of ALC-0315 LNPs (1:1 ratio, pre-incubated overnight with human serum) at the indicated doses for each condition in triplicate wells. The tested doses for each mRNA:gRNA pair (Table 70) were 2400, 800, 270, 90, 30, 10, 0.1, and 0.01 ng total encapsulated RNA per well.

Cells were maintained in Geltrex-sandwich culture and PCSK9 secretion was analyzed by HTRF ELISA in culture supernatants collected on day 5 after treatment using the CISBio Human PCSK9 HTRF ELISA Kit (Revvity). The cell culture experiment was repeated for 3 rounds.

For rounds 1 and 2, cells were immediately lysed in 100 μL DNA RNA shield ^TM and the media was collected on day 5 and stored at -80°C.

The original CasX 515 with targeting sequence 6.1 and LTRP5 with targeting sequence 27.94 were included on each plate as control treatments to assess the stability of the measurements throughout the experiment. qPCR analysis of PCSK9 mRNA attenuation:

Extracted mRNA was processed and analyzed by RT-qPCR. PCSK9 mRNA expression was measured using a single-step RT-qPCR method. 50 μL of lysate was used to extract total RNA using the Zymo 96 RNA extraction kit. Total RNA was then reverse transcribed into cDNA and PCSK9 transcripts were quantified by quantitative PCR relative to a GAPDH endogenous control using TaqMan probes. Percent change in mRNA was calculated compared to non-targeted sequence treated samples. ELISA:

The secreted PCSK9 levels in the culture supernatant were analyzed using the BioLegend® ELISA MAX ^™ Kit according to the manufacturer's instructions. RNA seq:

mRNA levels were analyzed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) according to the manufacturer's instructions. RNA from the highest dose samples treated with the specified mRNA-gRNA pairs in all wells (N=2 to 6 wells/condition) and the initial wells (N=4) was analyzed using double-end short-read RNA sequencing. Changes in gene expression relative to the initial cells were calculated from read counts using DEseq2. The cutoff values used were |log2(fold change)|>1 and Benjamini and Hochberg-adjusted p value (padj)<0.05. Results:

mRNA:gRNA pairs comprising scaffold 316 and a sequence targeting PCSK9 were evaluated for their ability to reduce PCSK9 secretion and reduce PCSK9 mRNA. Tables 71 to 74 below provide the half maximal inhibitory concentration (IC ₅₀ ), 90% maximal inhibition (IC ₉₀ ), and maximum response (Emax) in primary human hepatocytes using each individual mRNA:sgRNA pair, rounded to the nearest hundredth. Tables 71 and 72 provide the results of PCSK9 secretion reduction in serum ELISA dose response assays in ng encapsulated RNA/well and pM encapsulated RNA, respectively. Tables 73 and 74 provide the results of PCSK9 mRNA reduction in qPCR dose response assays in ng encapsulated RNA/well and pM encapsulated RNA, respectively.

LTRP5 and LTRP6-rat-cobra were the most potent molecules in reducing secreted proteins, and their reduction in secreted PCSK9 protein was similar to that of the positive control editors CasX 515 and SpyCas9, with 6.154 being the most potent LTRP spacer (Tables 71 and 72).

LTRP5 and LTRP6-rat-cobra were the most potent molecules in reducing PCSK9 mRNA levels, with reductions similar to those of the positive control editors CasX 515 and SpyCas9, with 6.154 being the most potent LTRP spacer (Tables 73 and 74).

The LTRP constructs LTRP6-rat-cobra, LTRP6-rat-cobra-linker set 3, and LTRP6-2×cobra paired with gRNA 316.6.154 (V1) showed the highest specificity as measured by changes in mRNA expression (Table 75). LTRP6-rat-cobra-linker set 3 and LTRP6-2×cobra had no significant off-target effects as measured by a padj cutoff value of <0.05.

These experimental results indicate that delivery of LNPs encapsulating LTRP mRNA and PCSK9 targeting gRNA induces inhibition at the endogenous human PCSK9 locus in primary human hepatocytes, thereby causing a substantial decrease in secreted PCSK9 and mRNA levels. Table 69 : Sequences of mRNA and gRNA mRNA SEQ ID NO. gRNA Target SEQ ID NO. LTRP5 22829 316.6.1 (V1) PCSK9 22789 LTRP6-2x Cobra 22830 316.6.154 (V1) PCSK9 22790 LTRP6-rat-cobra 22831 316.6.157 (V1) PCSK9 22788 LTRP6-rat-cobra-linker set 3 22832 316.6.141 (V1) PCSK9 22835 CasX 515 22833 316.27.94 (V1) NT 3330 SpyCas9 22834 Spy.50.1 (vInt) PCSK9 22791 Table 70 : mRNA:gRNA pairs tested Structure gRNA CasX 515 316.6.1 (V1) LTRP5 316.6.141 (V1) LTRP5 316.6.154 (V1) LTRP5 316.6.157 (V1) LTRP5 316.6.154 (V1), 316.6.157 (V1) LTRP5 316.27.94 (V1) LTRP6-2x Cobra 316.6.141 (V1) LTRP6-2x Cobra 316.6.154 (V1) LTRP6-2x Cobra 316.6.157 (V1) LTRP6-rat-cobra 316.6.141 (V1) LTRP6-rat-cobra 316.6.154 (V1) LTRP6-rat-cobra 316.6.157 (V1) LTRP6-rat-cobra-linker set 3 316.6.141 (V1) LTRP6-rat-cobra-linker set 3 316.6.154 (V1) LTRP6-rat-cobra-linker set 3 316.6.157 (V1) SpyCas9 Spy.50.1 (vInt) Table 71 : Levels of PCSK9 secretion inhibition mediated by LTRP constructs with various inhibitory subdomains as measured by total cargo mass PHH Batch 271 IC50 (ng/ well) IC90* (ng/ well) Emax* (Δ%) mRNA gRNA average value SEM average value SEM average value SEM CasX 515 316.6.1 (V1) 27.7 5 190.1 84.2 -94.6 4 LTRP5 316.6.154 (V1) 40 4 119.9 27.1 -98.8 3.1 SpyCas9 Spy.50.1 (vInt) 39.5 3.8 226.6 50.9 -99.1 2.8 LTRP5 316.6.154 (V1), 316.6.157 (V1) 42.6 4.6 114.5 26.5 -97.8 3.6 LTRP6-rat-cobra 316.6.154 (V1) 57.4 5.4 217.5 43.6 -98.9 2.7 LTRP5 316.6.141 (V1) 79.9 6.3 289.7 52.8 -97.2 2.5 LTRP6-2x Cobra 316.6.154 (V1) 81.8 9.2 286.6 74.6 -96.3 3.8 LTRP5 316.6.157 (V1) 96 10 491.6 122.3 -100.8 4 LTRP6-2x Cobra 316.6.141 (V1) 115.8 21.4 494.7 215.8 -99.5 7 LTRP6-rat-cobra 316.6.141 (V1) 131.5 14.8 552.5 146.2 -100.8 4.4 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 133.3 22.8 624 252.8 -98.3 5.9 LTRP6-rat-cobra 316.6.157 (V1) 177.6 38 1216.6 635 -99 8.6 LTRP6-2x Cobra 316.6.157 (V1) 173.3 37.7 1054.8 558.8 -91.5 8.5 LTRP6-rat-cobra-linker set 3 316.6.141 (V1) 247 56.6 933.6 530.2 -92.7 9.4 LTRP6-rat-cobra-linker set 3 316.6.157 (V1) 253.4 66.9 1195.8 780.8 -69.2 8.4 mRNA gRNA PHH Batch 31 CasX 515 316.6.1 (V1) 23.2 5.4 180 107.8 -93.2 5.2 LTRP5 316.6.154 (V1) 13.7 1.9 73.4 25.5 -97.4 3.4 SpyCas9 Spy.50.1 (vInt) 24.8 5.1 244.2 134.2 -100.1 5.7 LTRP5 316.6.154 (V1), 316.6.157 (V1) 29.1 2.3 80.1 15.6 -97.2 2.7 LTRP6-rat-cobra 316.6.154 (V1) 24.4 4.1 153.9 63.4 -100.7 4.6 LTRP5 316.6.141 (V1) 49.6 7.2 364.6 126.9 -100.6 4.1 LTRP6-2x Cobra 316.6.154 (V1) 72 16.6 327.5 171.8 -100.5 8 LTRP5 316.6.157 (V1) 58 8.1 222.9 66.9 -96.2 4.9 LTRP6-2x Cobra 316.6.141 (V1) 98.6 12.7 310 98 -98.8 5.8 LTRP6-rat-cobra 316.6.141 (V1) 89.4 9.3 284.2 71.6 -98.5 4.2 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 88.8 18.6 406.6 199.3 -96.2 7.1 LTRP6-rat-cobra 316.6.157 (V1) 101.3 18.1 634 271.4 -98.5 7 LTRP6-2x Cobra 316.6.157 (V1) 181.6 36.2 718.8 335.1 -92.5 9.4 LTRP6-rat-cobra-linker set 3 316.6.141 (V1) 158.9 29.9 527 218 -89.1 7.8 LTRP6-rat-cobra-linker set 3 316.6.157 (V1) 261.2 77.3 997.8 738.1 -72.3 12.7 Table 72 : PCSK9 secretion protein inhibition levels mediated by LTRP constructs with various inhibitory subdomains as measured by mRNA molar concentrations PHH Batch 271 IC50 (pM) IC90* (pM) Emax (Δ%) mRNA gRNA average value SEM average value SEM average value SEM LTRP5 316.6.154 (V1) 110.4 11.2 330.7 74.9 -98.8 3.1 LTRP5 316.6.154 (V1), 316.6.157 (V1) 117.6 12.6 315.8 73 -97.8 3.6 SpyCas9 Spy.50.1 (vInt) 128.1 12.5 734.7 165.1 -99.1 2.8 LTRP6-rat-cobra 316.6.154 (V1) 153.8 14.4 582.4 116.8 -98.9 2.7 CasX 515 316.6.1 (V1) 138.1 25.1 947 419.5 -94.6 4 LTRP5 316.6.141 (V1) 220.4 17.5 798.8 145.5 -97.2 2.5 LTRP6-2x Cobra 316.6.154 (V1) 219 24.6 767.7 199.8 -96.3 3.8 LTRP5 316.6.157 (V1) 264.6 27.6 1355.5 337.3 -100.8 4 LTRP6-2x Cobra 316.6.141 (V1) 310 57.3 1325.1 578 -99.5 7 LTRP6-rat-cobra 316.6.141 (V1) 352.1 39.7 1479.3 391.5 -100.8 4.4 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 365.9 62.5 1713.1 694 -98.3 5.9 LTRP6-rat-cobra 316.6.157 (V1) 475.6 101.7 3257.6 1700.3 -99 8.6 LTRP6-2x Cobra 316.6.157 (V1) 464.2 101 2825 1496.7 -91.5 8.5 LTRP6-rat-cobra-linker set 3 316.6.141 (V1) 678.1 155.5 2563 1455.4 -92.7 9.4 LTRP6-rat-cobra-linker set 3 316.6.157 (V1) 695.8 183.7 3282.8 2143.6 -69.2 8.4 mRNA gRNA PHH Batch 31 LTRP5 316.6.154 (V1) 37.9 5.2 202.4 70.3 -97.4 3.4 LTRP5 316.6.154 (V1), 316.6.157 (V1) 80.2 6.4 221 42.9 -97.2 2.7 SpyCas9 Spy.50.1 (vInt) 80.5 16.6 791.8 435.2 -100.1 5.7 LTRP6-rat-cobra 316.6.154 (V1) 65.2 11.1 412.2 169.7 -100.7 4.6 CasX 515 316.6.1 (V1) 115.5 27.1 896.6 537 -93.2 5.2 LTRP5 316.6.141 (V1) 136.8 19.9 1005.3 350 -100.6 4.1 LTRP6-2x Cobra 316.6.154 (V1) 192.7 44.4 877.1 460.3 -100.5 8 LTRP5 316.6.157 (V1) 160.1 22.3 614.7 184.5 -96.2 4.9 LTRP6-2x Cobra 316.6.141 (V1) 264 34 830.3 262.5 -98.8 5.8 LTRP6-rat-cobra 316.6.141 (V1) 239.3 24.9 761 191.6 -98.5 4.2 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 243.9 51.1 1116.2 547.1 -96.2 7.1 LTRP6-rat-cobra 316.6.157 (V1) 271.3 48.5 1697.7 726.8 -98.5 7 LTRP6-2x Cobra 316.6.157 (V1) 486.2 97 1925.3 897.6 -92.5 9.4 LTRP6-rat-cobra-linker set 3 316.6.141 (V1) 436.2 82.1 1446.8 598.5 -89.1 7.8 LTRP6-rat-cobra-linker set 3 316.6.157 (V1) 717.1 212.2 2739.2 2026.2 -72.3 12.7 Table 73 : PCSK9 mRNA inhibition levels mediated by LTRP constructs with various inhibitory subdomains as measured by total cargo PHH Batch 271 IC50 (ng/ well) IC90* (ng/ well) Emax* (Δ%) mRNA gRNA average value SEM average value SEM average value SEM CasX 515 316.6.1 (V1) 29.6 5.7 103.3 45.2 -60.1 2.7 SpyCas9 Spy.50.1 (vInt) 50.3 8.2 161.2 53.9 -59.8 2.6 LTRP5 316.6.154 (V1) 38.2 7.8 211.2 101 -96.7 5.5 LTRP5 316.6.154 (V1), 316.6.157 (V1) 45.7 4.5 143.1 30.4 -95.1 2.9 LTRP6-rat-cobra 316.6.154 (V1) 92.9 13.7 357.9 124.5 -94.9 4.3 LTRP5 316.6.157 (V1) 101.3 14.5 504.4 173.5 -94.7 5 LTRP6-2x Cobra 316.6.154 (V1) 123.7 19.8 528.3 199.1 -91.9 5 LTRP5 316.6.141 (V1) 112.8 13.8 354.6 104.1 -88.9 3.7 LTRP6-rat-cobra 316.6.141 (V1) 153.1 22.4 755 264.3 -92.9 4.5 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 168.6 36 793.8 405.5 -83.3 6.4 LTRP6-2x Cobra 316.6.141 (V1) 159.7 47.4 695.8 486.5 -92.7 8.7 LTRP6-rat-cobra 316.6.157 (V1) 182.7 35.7 957 453.1 -80.3 5.8 LTRP6-2x Cobra 316.6.157 (V1) 189.8 42.8 817.3 439.1 -70.7 6.2 LTRP6-rat-cobra-linker set 3 316.6.157 (V1) 135.1 37.9 470.1 300.4 -41.1 4.3 LTRP6-rat-cobra-linker set 3 316.6.141 (V1) 233.2 67.6 1054.7 750.9 -73.1 8.1 PHH Batch 31 mRNA gRNA average value SEM average value SEM average value SEM CasX 515 316.6.1 (V1) 42.7 10.6 149.3 80.3 -54.4 3.3 SpyCas9 Spy.50.1 (vInt) 37.5 28.2 293.3 539.3 -44.4 9.4 LTRP5 316.6.154 (V1) 53.8 10.9 202.5 87.7 -97 4.6 LTRP5 316.6.154 (V1), 316.6.157 (V1) 59.7 9.2 194.2 61.6 -96.5 3.9 LTRP6-rat-cobra 316.6.154 (V1) 39.9 5.7 387 139.8 -96.1 3.6 LTRP5 316.6.157 (V1) 115.9 10.6 438.4 93.9 -93.4 2.5 LTRP6-2x Cobra 316.6.154 (V1) 106.8 16.8 327.2 125.7 -89.6 5.2 LTRP5 316.6.141 (V1) 122 30.2 701.4 417.1 -92.1 7.3 LTRP6-rat-cobra 316.6.141 (V1) 115 13.2 407.9 110.7 -88.6 3.6 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 129.6 22.2 461.3 182.3 -83.1 5.2 LTRP6-2x Cobra 316.6.141 (V1) 148.6 18.7 377.4 93.9 -89.7 4.1 LTRP6-rat-cobra 316.6.157 (V1) 172.1 42.4 1053.3 631.5 -87.3 8 LTRP6-2x Cobra 316.6.157 (V1) 196.7 35.5 659.9 271.6 -77.4 6 LTRP6-rat-cobra-linker set 3 316.6.157 (V1) 254.2 69.8 728.3 523.5 -55.4 7.6 LTRP6-rat-cobra-linker set 3 316.6.141 (V1) 228 37 536.7 210 -73.7 5.4 Table 74 : PCSK9 mRNA inhibition levels mediated by LTRP constructs with various inhibitory subdomains as measured by mRNA molar concentration PHH Batch 271 IC50 (pM) IC90* (pM) Emax* (Δ%) mRNA gRNA average value SEM average value SEM average value SEM LTRP5 316.6.154 (V1) 105.3 21.4 582.3 278.4 -96.7 5.5 SpyCas9 Spy.50.1 (vInt) 163.2 26.6 522.5 174.8 -59.8 2.6 LTRP5 316.6.154 (V1), 316.6.157 (V1) 126 12.5 394.7 83.8 -95.1 2.9 LTRP6-rat-cobra 316.6.154 (V1) 248.7 36.7 958.4 333.5 -94.9 4.3 CasX 515 316.6.1 (V1) 147.5 28.6 514.9 225 -60.1 2.7 LTRP5 316.6.154 (V1) 279.3 40 1390.8 478.5 -94.7 5 LTRP6-2x Cobra 316.6.154 (V1) 331.4 53 1415.1 533.1 -91.9 5 LTRP5 316.6.154 (V1) 311 38.1 977.8 286.9 -88.9 3.7 LTRP6-rat-cobra 316.6.154 (V1) 409.9 60 2021.7 707.6 -92.9 4.5 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 462.8 99 2179.2 1113.3 -83.3 6.4 LTRP6-2x Cobra 316.6.154 (V1) 427.7 126.9 1863.5 1303 -92.7 8.7 LTRP6-rat-cobra 316.6.154 (V1) 489.3 95.5 2562.4 1213.2 -80.3 5.8 LTRP6-2x Cobra 316.6.154 (V1) 508.4 114.6 2188.9 1175.9 -70.7 6.2 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 370.8 103.9 1290.5 824.5 -41.1 4.3 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 640.1 185.5 2895.5 2061.3 -73.1 8.1 PHH Batch 31 mRNA gRNA average value SEM average value SEM average value SEM LTRP5 316.6.154 (V1) 148.3 30.1 558.3 241.9 -97 4.6 SpyCas9 Spy.50.1 (vInt) 121.5 91.3 950.9 1748.5 -44.4 9.4 LTRP5 316.6.154 (V1), 316.6.157 (V1) 164.6 25.3 535.5 169.9 -96.5 3.9 LTRP6-rat-cobra 316.6.154 (V1) 106.8 15.2 1036.2 374.3 -96.1 3.6 CasX 515 316.6.1 (V1) 212.8 52.8 743.7 399.9 -54.4 3.3 LTRP5 316.6.154 (V1) 319.5 29.1 1208.7 259 -93.4 2.5 LTRP6-2x Cobra 316.6.154 (V1) 286.1 44.9 876.3 336.5 -89.6 5.2 LTRP5 316.6.154 (V1) 336.3 83.3 1934.1 1150.2 -92.1 7.3 LTRP6-rat-cobra 316.6.154 (V1) 307.8 35.5 1092.1 296.5 -88.6 3.6 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 355.7 61 1266.3 500.4 -83.1 5.2 LTRP6-2x Cobra 316.6.154 (V1) 398 50 1010.9 251.4 -89.7 4.1 LTRP6-rat-cobra 316.6.154 (V1) 460.9 113.4 2820.4 1690.8 -87.3 8 LTRP6-2x Cobra 316.6.154 (V1) 526.9 95.2 1767.5 727.6 -77.4 6 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 697.9 191.7 1999.3 1437.2 -55.4 7.6 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 625.9 101.5 1473.4 576.4 -73.7 5.4 Table 75 : Gene expression changes determined by RNA-seq mRNA gRNA Dose(ng*) Gene log2( fold change) P- value CasX 515 316.6.1 (V1) 2400 TUBB3 -1.05 5.90E-08 MARCO -2.67 8.61E-07 ESAM -1.04 1.07E-06 SUSD2 -1.14 3.07E-06 Q1Q -1.67 2.87E-05 KIF23 -1.19 5.82E-05 SpyCas9 Spy.50.1 (vInt) 2400 PLD4 -1.36 4.14E-07 SLC7A7 -1.03 8.09E-07 TSPAN15 -1.28 1.54E-06 ANXA8 -1.28 3.39E-06 RAC2 -1.08 4.94E-06 CYP7A1 1.45 1.09E-05 LTRP5 316.6.154 (V1), 316.6.157 (V1) 2400 PLD4 -1.36 4.14E-07 SLC7A7 -1.03 8.09E-07 TSPAN15 -1.28 1.54E-06 ANXA8 -1.28 3.39E-06 RAC2 -1.08 4.94E-06 CYP7A1 1.45 1.09E-05 LTRP5 316.6.154 (V1) 2400 PCSK9 -3.87 1.93E-64 Q1Q -2.92 1.78E-08 GPNMB -1.96 3.06E-08 CD163 -1.49 3.89E-07 ITGAX -2.02 5.74E-07 LAPTM5 -1.60 1.33E-06 RNASE1 -2.12 2.05E-06 MSR1 -2.30 2.10E-06 RGCC -2.20 2.88E-06 PLEK -2.46 1.88E-05 MIR6501 1.04 0.00011389 HCK -2.84 0.00013665 LTRP5 316.6.157 (V1) 2400 PCSK9 -2.97 9.32E-41 TM4SF19 -2.59 1.26E-06 UCP2 -2.07 3.00E-05 MIR6501 1.09 8.01E-05 LTRP5 316.27.94 (V1) 2400 NA NA NA LTRP6-rat-cobra 316.6.154 (V1) 2400 PCSK9 -3.34 5.86E-47 DNMT3A 1.83 1.55E-08 G6PD 1.01 4.75E-05 SDS 1.47 0.00012577 LTRP6-rat-cobra-linker set 3 316.6.154 (V1) 2400 PCSK9 -1.45 3.00E-12 LTRP6-2x Cobra 316.6.154 (V1) 2400 PCSK9 -2.34 2.85E-27 *Dose is reported as ng encapsulated RNA/well

The following figures will be referenced to facilitate understanding of the features and advantages of the present disclosure:

Figure 1 shows schematic diagrams of various configurations of inhibitor fusion proteins incorporating the DNMT3A ADD domain. "D3A ADD", "D3A CD" and "D3L ID" represent the ADD domain of DNMT3A, the catalytic domain of DNMT3A and the interaction domain of DNMT3L, respectively. L1 to L3 are linkers. NLS is a nuclear localization signal.

Figure 2 is a bar graph showing quantification of secreted PCSK9 levels at 6, 18, and 36 days post-transfection in Huh7 cells transfected with mRNA liposomes encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 paired with the indicated targeting gRNAs, as described in Example 1. Secreted PCSK9 levels were normalized to total cell counts. Naïve, untreated cells were used as experimental controls.

Figure 3 is a bar graph showing quantification of normalized secreted PCSK9 levels at day 4 post-transfection in HepG2 cells transfected with mRNA liposomes encoding CasX 676, dXR1 or LTRP5-ADD-ZIM3 paired with a specified targeting gRNA, as described in Example 2. Secreted PCSK9 levels were normalized to total cell counts. Initial untreated cells were used as experimental controls.

Figure 4 is a bar graph showing quantification of normalized secreted PCSK9 levels 4 days after transfection in Huh7 cells transfected with mRNA liposomes encoding CasX 676, dXR1 or LTRP5-ADD-ZIM3 paired with the specified targeting gRNA, as described in Example 2. Secreted PCSK9 levels were normalized to total cell counts. Initial untreated cells were used as experimental controls.

Figure 5 is a bar graph showing quantification of normalized secreted PCSK9 levels 4 days after transfection in Hep3B cells transfected with mRNA liposomes encoding CasX 676, dXR1 or LTRP5-ADD-ZIM3 paired with the specified targeting gRNA, as described in Example 2. Secreted PCSK9 levels were normalized to total cell counts. Initial untreated cells were used as experimental controls.

Figure 6 is a bar graph showing quantification of secreted PCSK9 levels at 4, 14 and 27 days post-transfection in Huh7 cells transfected with mRNA liposomes encoding CasX 676, dXR1 or LTRP5-ADD-ZIM3 paired with the indicated targeting gRNAs, as described in Example 2. Quantification of secreted PCSK9 levels is shown relative to the secretion levels detected in the initial, untreated cells at the 4 day time point.

Figure 7 is a schematic diagram of gRNA scaffold variant 174 (SEQ ID NO: 1744) as described in Example 3. The structural motif is highlighted.

FIG8 is a schematic diagram of gRNA scaffold variant 235 (SEQ ID NO: 1745) as described in Example 3. The highlighted structural motifs are the same as in FIG7. The differences between variant 174 and variant 235 are in the extension stem motif and several single nucleotide changes (indicated by asterisks). Variant 316 maintains the shorter extension stem from variant 174, but has four substitutions found in scaffold 235.

Figure 9 is a schematic diagram of gRNA scaffold variant 316 (SEQ ID NO: 1746) as described in Example 3. The highlighted structural motifs are the same as in Figure 7. Variant 316 maintains the shorter extended stem from variant 174 (Figure 7), but has four substitutions found in scaffold 235 (Figure 8).

FIG. 10 is a schematic diagram showing versions 1 to 3 of chemical modifications to gRNA scaffold variant 235 as described in Example 3. The structural motifs are highlighted. Standard ribonucleotides are depicted as open circles and 2'OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with an * below or next to the bond. For the v2 profile, the addition of three 3' uracils (3'UUU) is indicated with a "U" in the associated circle.

FIG. 11 is a schematic diagram showing versions 4 to 6 of chemical modifications of gRNA scaffold variant 235 as described in Example 3. The structural motif is highlighted. Standard ribonucleotides are depicted as open circles and 2'OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or next to the bond.

FIG. 12 is a schematic diagram showing versions 7 to 9 of chemical modifications of gRNA scaffold variant 316 as described in Example 3. The structural motif is highlighted. Standard ribonucleotides are depicted as open circles and 2'OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or next to the bond.

FIG. 13 is a schematic diagram showing patterns 1 to 3 of chemical modifications to gRNA scaffold variant 316 as described in Example 3. The structural motif is highlighted. Standard ribonucleotides are depicted as open circles and 2'OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or next to the bond.

FIG. 14 is a schematic diagram showing versions 4 to 6 of chemical modifications of gRNA scaffold variant 316 as described in Example 3. The structural motif is highlighted. Standard ribonucleotides are depicted as open circles and 2'OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or next to the bond.

15 is a graph showing the correlation between the insertion/deletion rate (depicted as edited portions) of the PCSK9 locus measured by next generation sequencing (NGS) (x-axis) and the secreted PCSK9 levels (ng/mL) detected by enzyme-linked immunosorbent assay (ELISA) (y-axis) in HepG2 cells transfected with CasX 491 mRNA and PCSK9 -targeting gRNA containing the specified scaffold variants and spacer combinations by liposomes, as described in Example 3.

16 is a graph showing quantification of the percentage of B2M gene knockout in HepG2 cells co-transfected with 100 ng of CasX 491 mRNA and indicated amounts of B2M targeting gRNAs with ends modified (v1) or unmodified (v0) with spacer 7.37, as described in Example 3. For cell populations that lost surface presentation of the HLA complex due to successful editing at the B2M locus, editing levels were determined by flow cytometry.

17 is a graph depicting the results of editing analysis measured as insertion/deletion rates detected by NGS at the human B2M locus in HepG2 cells treated with specified doses of LNPs formulated with CasX 491 mRNA and specified B2M- targeting gRNAs as described in Example 3.

Figure 18 is a graph showing the quantification of the percentage of B2M gene knockout in HepG2 cells treated with the indicated doses of LNPs formulated with CasX491 mRNA and the indicated B2M targeting gRNAs as described in Example 3. Editing levels were measured by flow cytometry for cell populations that did not have surface presentation of HLA complexes due to successful editing at the B2M locus.

Figure 19 is a graph depicting the results of editing analysis measured as insertion/deletion rates detected by NGS at the mouse ROSA26 locus in Hepa1-6 cells treated with the specified doses of LNPs formulated with CasX676 mRNA #2 and the specified ROSA26- targeting gRNAs with v1 or v5 modification profiles, as described in Example 3.

FIG. 20 is a graph showing the quantification of percent editing measured as the indel rate detected by NGS at the ROSA26 locus in mice treated with LNPs formulated with CasX 676 mRNA #2 and the indicated chemically modified ROSA26 targeting gRNAs as described in Example 3.

Figure 21 is a bar graph showing the results of an edit analysis measured as the insertion/deletion rate detected by NGS at the mouse PCSK9 locus in mice treated with LNPs formulated with CasX676 mRNA #1 and the indicated chemically modified PCSK9 targeting gRNAs as described in Example 3. Untreated mice were used as experimental controls.

Figure 22 is a diagram of the secondary structure of guide RNA scaffold 235 (SEQ ID NO: 1745), indicating regions with CpG motifs, as described in Example 6. CpG motifs in (1) pseudostem, (2) scaffold stem, (3) extension stem vesicle, (4) extension step, and (5) extension stem loop are marked on the structure.

FIG. 23 is a diagram of CpG reduction mutations introduced into each of the five regions in the coding sequence of the guide RNA scaffold, as described in Example 6.

FIG. 24 provides the results of an editing experiment in which AAV vectors with various CpG-reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. AAV vectors were administered at a multiplicity of infection (MOI) of 4e3. Bars show the mean ± SD of two replicates per sample. "No Tx" indicates a control that was not transduced, and "NT" indicates a control with a non-targeting spacer.

Figure 25 provides the results of an editing experiment in which AAV vectors with various CpG-reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. The AAV vectors were administered at an MOI of 3e3. The bars show the mean ± SD of two replicates per sample. "Untreated" indicates a non-transduced control.

Figure 26 provides the results of an editing experiment in which AAV vectors with various CpG-reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. The AAV vectors were administered at an MOI of 1e3. The bars show the mean ± SD of two replicates per sample. "Untreated" indicates a non-transduced control.

Figure 27 provides the results of an editing experiment in which AAV vectors with various CpG-reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. The AAV vectors were administered at an MOI of MOI = 3e2. The bars show the mean ± SD of two replicates per sample. "Untreated" indicates a non-transduced control.

Figure 28 shows schematic diagrams of five configurations of long-term repressor protein (LTRP, also referred to herein as "repressor fusion protein") fusion proteins, in which the repressor molecule is linked to a catalytically inactive CasX. D3A and D3L represent DNA methyltransferase 3α (DNMT3A) and DNMT3A-like protein (DNMT3L), respectively. L1 to L4 are linkers. NLS is a nuclear localization signal.

Figure 29 shows schematics of various LTRP #5 architectures in which additional DNMT3A domains are incorporated, as described in Example 7. The additional DNMT3A domains are the ADD domain of DNMT3A ("D3A ADD") and the PWWP domain (Pro-Trp-Trp-Pro motif) of DNMT3A ("D3A PWWP"). "D3A endo" encodes the endogenous sequence that occurs between the DNMT3A PWWP and ADD domains. "D3A CD" and "D3L ID" represent the catalytic domain of DNMT3A and the interaction domain of DNMT3L, respectively. "L1 to L3" is a linker. "NLS" is a nuclear localization signal. See Table 13 for LTRP sequences.

Figure 30 presents the results of a time course experiment comparing the B2M inhibitory activity (expressed as the percentage of HLA-negative cells) of the indicated LTRP-ZIM3 and its variants with B2M -targeting gRNA using spacer 7.37, as described in Example 7. Data are presented as means with standard deviations, N = 3. CD = catalytic domain of DNMT3A.

Figure 31 presents the results of the same time course experiment shown in Figure 30, but showing the B2M inhibitory activity of the indicated LTRP-ZIM3 variants with B2M targeting gRNA using spacer 7.160, as described in Example 7. Data are presented as means with standard deviations, N=3.

Figure 32 presents the results of the same time course experiment shown in Figure 30, but showing the B2M inhibitory activity of the indicated LTRP-ZIM3 variants with B2M targeting gRNA using spacer 7.165, as described in Example 7. Data are presented as means with standard deviations, N=3.

Figure 33 presents the results of the same time course experiment shown in Figure 30, but showing the B2M inhibitory activity of the indicated LTRP-ZIM3 variants with non-targeting gRNA, as described in Example 7. Data are presented as means with standard deviations, N=3.

FIG. 34 is a violin plot of the CpG methylation percentage of the CpG sites downstream of the transcription start site of the VEGFA locus of each designated LTRP-ZIM3 variant for three B2M- targeting gRNAs and non-targeting gRNAs, as described in Example 7.

Figure 35 is a scatter plot showing the relative activity (average percentage of HLA-negative cells on day 21 for spacer 7.160) and specificity (percent off-target CpG methylation at the VEGFA locus quantified on day 7 for spacer 7.160) of the indicated LTRP5-ZIM3 variants, as described in Example 7.

FIG36 is a schematic diagram showing the general architecture of LTRP molecules with ADD domains for LTRP configurations #1, #4, and #5 tested in Example 8. "D3A ADD", "D3A CD", and "D3L ID" represent the ADD domain of DNMT3A, the catalytic domain of DNMT3A, and the interaction domain of DNMT3L, respectively, as described in Example 8. "L1 to L4" is a linker. "NLS" is a nuclear localization signal. See Table 11 for LTRP sequences.

Figure 37 presents the results of a time course experiment comparing the B2M inhibitory activity (expressed as the percentage of HLA-negative cells) of LTRPs with ZIM3-KRAB domain, with configuration #1, #4 or #5, with or without DNMT3A ADD domain, paired with B2M targeting gRNA with spacer 7.160, as described in Example 8. Data are presented as means with standard deviations, N = 3. "NT" is a gRNA with a non-targeting spacer.

Figure 38 is a graph showing the results of the same time course experiments shown in Figure 37, but showing the B2M inhibitory activity of LTRP #5 with ZNF10 or ZIM3-KRAB domains, with or without DNMT3A ADD domain, paired with B2M targeting gRNA with spacer 7.160, as described in Example 8. Data are presented as means with standard deviations, N = 3. "NT" is gRNA with a non-targeting spacer.

Figure 39 is a graph showing the results of the same time course experiment shown in Figure 37, but showing the B2M inhibitory activity of LTRP5-ZIM3 with or without the DNMT3A ADD domain, paired with a B2M targeting gRNA with the specified spacer, as described in Example 8. Data are presented as means with standard deviations, N = 3. "NT" is a gRNA with a non-targeting spacer.

Figure 40 is a graph showing the results of B2M inhibitory activity at day 27 post-transfection for LTRPs with ZNF10 or ZIM3-KRAB domains, with configuration #1, with or without DNMT3A ADD domains for the indicated gRNAs, as described in Example 8. Data are presented as means with standard deviations, N = 3. "NT" is a gRNA with a non-targeting spacer.

Figure 41 is a graph showing the results of B2M inhibitory activity of LTRPs with ZNF10 or ZIM3-KRAB domains, with configuration #4, with or without DNMT3A ADD domain at day 27 post-transfection for the indicated gRNAs, as described in Example 8. Data are presented as mean with standard deviation, N = 3. "NT" is a gRNA with a non-targeting spacer.

Figure 42 is a graph showing the results of B2M inhibitory activity of LTRPs with ZNF10 or ZIM3-KRAB domains, with configuration #5, with or without DNMT3A ADD domain at day 27 post-transfection for the indicated gRNAs, as described in Example 8. Data are presented as mean with standard deviation, N = 3. "NT" is a gRNA with a non-targeting spacer.

Figure 43 is a graph showing the results of bisulfite sequencing of LTRPs with ZNF10 or ZIM3-KRAB domains, with configuration #1, with or without DNMT3A ADD domain, for the indicated gRNAs at day 5 post-transfection to determine off-target methylation at the VEGFA locus, as described in Example 8. Data are presented as the average percentage of CpG methylation at CpG sites near the VEGFA locus; standard error of the mean is also presented; N = 3. "NT" is a gRNA with a non-targeting spacer.

Figure 44 is a graph showing the results of bisulfite sequencing of LTRPs with ZNF10 or ZIM3-KRAB domains, with configuration #4, with or without DNMT3A ADD domain, for the indicated gRNAs at day 5 post-transfection to determine off-target methylation at the VEGFA locus, as described in Example 8. Data are presented as the average percentage of CpG methylation at CpG sites near the VEGFA locus; standard error of the mean is also presented; N = 3. "NT" is a gRNA with a non-targeting spacer.

Figure 45 is a graph showing the results of bisulfite sequencing of LTRPs with ZNF10 or ZIM3-KRAB domains, with configuration #5, with or without DNMT3A ADD domain, for the indicated gRNAs at day 5 post-transfection to determine off-target methylation at the VEGFA locus, as described in Example 8. Data are presented as the average percentage of CpG methylation at CpG sites near the VEGFA locus; standard error of the mean is also presented; N = 3. "NT" is a gRNA with a non-targeting spacer.

Figure 46 is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) and specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules with ZIM3-KRAB domain, configurations #1, #4 and #5 for B2M targeting gRNA with spacer 7.160, as described in Example 8.

Figure 47 is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) and specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules with ZNF10- KRAB domain, configurations #1, #4 and #5 for B2M targeting gRNA with spacer 7.160, as described in Example 8.

Figure 48 is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) and specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules with ZIM3-KRAB domain, configurations #1, #4 and #5 for B2M targeting gRNA with spacer 7.37, as described in Example 8.

Figure 49 is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) and specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules with ZNF10- KRAB domain, configurations #1, #4 and #5 for B2M targeting gRNA with spacer 7.37, as described in Example 8.

Figure 50 is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) and specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules with ZIM3-KRAB domain, configurations #1, #4 and #5 for B2M targeting gRNA with spacer 7.165, as described in Example 8.

Figure 51 is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) and specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules with ZNF10- KRAB domain, configurations #1, #4 and #5 for B2M targeting gRNA with spacer 7.165, as described in Example 8.

52 is a graph showing the percentage of HEK293T cells transfected with plasmids encoding the indicated CasX or LTRP:gRNA constructs expressing B2M six days after treatment with different concentrations of the DNMT1 inhibitor 5-azadC, as described in Example 9.

53 is a graph juxtaposing quantification of B2M inhibition in HEK293T cells transfected with plasmids encoding the indicated CasX or LTRP:gRNA constructs and cultured for 58 days, and quantification of B2M reactivation following treatment of the transfected cells with 5-azadC, as described in Example 9.

Figure 54 shows a schematic diagram of an LTRP5 molecule without the ADD domain of DNMT3A as described in Example 11. "D3A CD" and "D3L ID" represent the catalytic domain of DNMT3A and the interaction domain of DNMT3L, respectively. "L1", "L2", "L3A" and "L3B" are linkers. "NLS" is a nuclear localization signal. "RD1" represents the inhibitory subdomain.

Figure 55 is a bar graph showing the results of a time course experiment comparing the level of B2M inhibition (expressed as the average percentage of HLA-negative cells) in HEK293T cells transfected with LTRP5 variant plasmids containing linker subsets 1-11, as described in Example 13. Data for each time point (Day 8, Day 15, and Day 45) are superimposed and presented as mean plus standard deviation, N = 3. A non-targeting spacer (NT) was included as an experimental control.

Figure 56 is a bar graph showing the results of a time course experiment comparing the levels of Target 1 inhibition (expressed as the percentage of total cells with Target 1 attenuation) of LTRP5 variants containing linker set 1-11 measured in HEK293T, as described in Example 13. Data for each time point (Day 8, Day 15, and Day 45) are overlaid and presented as mean plus standard deviation, N=3.

Figure 57 is a bar graph showing the results of a time course experiment comparing the levels of Target 2 inhibition (expressed as a percentage of total cells with Target 2 attenuation) of LTRP5 variants containing linker set 1-11 measured in HEK293T, as described in Example 13. Data for each time point (Day 8, Day 15, and Day 45) are superimposed and presented as mean plus standard deviation, N = 3. A non-targeting spacer (NT) was included as an experimental control.

Figure 58 is a bar graph showing the results of a time course experiment comparing the levels of B2M inhibition (expressed as the average percentage of HLA-negative cells) in HEK293T cells transfected with LTRP5 variant plasmids containing linker subsets 12-28, as described in Example 13. Data for each time point (day 7 and day 17) are superimposed and presented as mean plus standard deviation, N = 3. A non-targeting spacer (NT) was included as an experimental control. A non-targeting spacer (NT) was included as an experimental control.

Figure 59 is a bar graph showing the results of a time course experiment comparing the levels of Target 1 inhibition (expressed as a percentage of total cells with Target 1 attenuation) of LTRP5 variants containing linker subsets 12-28 measured in HEK293T, as described in Example 13. Data for each time point (day 7 and day 17) are superimposed and presented as mean plus standard deviation, N = 3. A non-targeting spacer (NT) was included as an experimental control.

Figure 60 is a bar graph showing the results of a time course experiment comparing the levels of Target2 inhibition (expressed as a percentage of total cells with Target2 attenuation) of LTRP5 variants containing linker subsets 12-28 measured in HEK293T, as described in Example 13. Data for each time point (day 7 and day 17) are superimposed and presented as mean plus standard deviation, N = 3. A non-targeting spacer (NT) was included as an experimental control.

Figure 61 shows schematic diagrams of various configurations of LTRP molecules with DNMT3A ADD domains. "D3A ADD", "D3A CD" and "D3L ID" represent the ADD domain of DNMT3A, the catalytic domain of DNMT3A and the interaction domain of DNMT3L, respectively. "L1", "L2", "L3A", "L3B" and "L4" are linkers. "NLS" is a nuclear localization signal. "RD1" represents an inhibitory subdomain, and "RD1a" and "RD1b" represent inhibitory subdomain variants.

Figure 62 is a violin plot where each point represents the average methylation% at an individual CpG. The median methylation is indicated by a dashed line, with the upper and lower quartiles indicated by dotted lines. DNA methylation near the transcription start site (TSS) was measured by amplicon enzymatic methylation sequencing (EM-seq) from homogenized gDNA extracted from the liver of N=3 mice sacrificed on days 7, 14, and 42 after treatment, as described in Example 14.

Figure 63 is a violin plot where each point represents the average methylation% at an individual CpG. The median methylation is indicated by a dashed line, with the upper and lower quartiles indicated by dotted lines. DNA methylation near the transcription start site (TSS) was measured by amplicon enzymatic methylation sequencing (EM-seq) from homogenized gDNA extracted from the liver of N=3 mice sacrificed on day 7 post-treatment, as described in Example 14.

TW202444906A_113111756_SEQL.xml

Claims (164)

A system for transcriptionally inhibiting the proprotein convertase subtilisin/kexin Type 9 ( PCSK9 ) gene, the system comprising: (a) a guide RNA (gRNA) comprising a targeting sequence complementary to the PCSK9 gene target nucleic acid sequence, and (b) an mRNA encoding a long-term suppressor fusion protein (LTRP), wherein the LTRP comprises: a. catalytically-dead CasX (dCasX); b. DNA methyltransferase (DNMT) 3A catalytic domain (DNMT3A); c. DNMT3-like interaction domain (DNMT3L); and d. a first suppressor domain (RD1), wherein the LTRP is capable of forming a ribonucleoprotein (RNP) with the gRNA. The system of claim 1, wherein the LTRP comprises from the N-terminus to the C-terminus: a.   The DNMT3A; b.   The DNMT3L; c.   The dCasX; and d.   The RD1. The system of claim 1, wherein the LTRP comprises from the N-terminus to the C-terminus: a.   The DNMT3A; b. The DNMT3L; c.   The RD1; and d.   The dCasX. A system as claimed in claim 3, wherein the LTRP further comprises another RD1 located at the C-terminus of the dCasX. A system as claimed in claim 4, wherein the RD1 sequences are identical. A system as claimed in claim 4, wherein the RD1 sequences are different. A system as claimed in any one of claims 1 to 6, wherein the LTRP comprises a DNA DNMT3A ATRX-DNMT3-DNMT3L domain (ADD) linked to the N-terminus of the DNMT3A. A system as claimed in any one of claims 1 to 7, wherein the LTRP comprises one or more linker peptides. The system of claim 8, wherein at least one of the one or more linker peptides comprises a sequence independently selected from the group consisting of SEQ ID NOs: 98 to 124 and 3278 to 3289. A system as claimed in any one of claims 1 to 9, wherein the LTRP comprises one or more nuclear localization signals (NLS). The system of claim 10, wherein at least one of the one or more NLSs comprises a sequence selected from the group consisting of SEQ ID NOs: 30 to 97. A system as claimed in claim 10 or claim 11, wherein at least one of the one or more NLSs is a Simian Virus 40 (SV40) NLS. The system of claim 12, wherein the SV40 NLS comprises the sequence of SEQ ID NO: 30. A system as claimed in claim 10 or claim 11, wherein at least one of the one or more NLSs is a c-MYC NLS. The system of claim 14, wherein the c-MYC NLS comprises the sequence of SEQ ID NO: 32. A system as claimed in any one of claims 1 to 2 or 8 to 15, wherein the LTRP comprises from N-terminus to C-terminus: a.   The first NLS; b.   The DNMT3A; c.   The first linker peptide; d.   The DNMT3L; e.   The second linker peptide; f.   The third linker peptide; g.   The dCasX; h.   The fourth linker peptide; i.   The RD1, and j.   The second NLS. The system of claim 16, wherein the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 22836 and SEQ ID NO: 22838 to 22846. A system as claimed in any one of claims 7 to 15, wherein the LTRP comprises from N-terminus to C-terminus: a.   The first NLS; b.   The ADD; c.   The DNMT3A; d.   The first linker peptide; e.   The DNMT3L; f.   The second linker peptide; g.   The third linker peptide; h.   The dCasX; i.   The fourth linker peptide; j.   The RD1, and k.   The second NLS. The system of claim 18, wherein the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 22837 and SEQ ID NO: 22847 to 22855. A system as claimed in any one of claims 1, 3 or 8 to 15, wherein the LTRP comprises from N-terminus to C-terminus: a.   The first NLS; b.   The DNMT3A; c.   The first linker peptide; d.   The DNMT3L; e.   The third linker peptide; f.   The RD1; g.   The second linker peptide; h.   The dCasX; i.   The fourth linker peptide, and j.   The second NLS. A system as claimed in any one of claims 7 to 15, wherein the LTRP comprises from N-terminus to C-terminus: a.   The first NLS; b.   The ADD; c.   The DNMT3A; d.   The first linker peptide; e.   The DNMT3L; f.   The second linker peptide; g.   The RD1 h.   The third linker peptide; i.   The dCasX; j.   The fourth linker peptide, and k.   The second NLS. A system as claimed in any one of claims 7 to 15, wherein the LTRP comprises from N-terminus to C-terminus: a.   The first NLS; b.   The ADD; c.   The DNMT3A; d.   The first linker peptide; e.   The DNMT3L; f.   The second linker peptide; g.   The RD1 h.   The third linker peptide; i.   The dCasX; j.   The fourth linker peptide; k.   Another RD1; l.   The fifth linker, and m.   The second NLS. A system as claimed in claim 22, wherein the RD1 sequence and the other RD1 sequence are identical. A system as claimed in claim 22, wherein the RD1 sequence and the other RD1 sequence are different. The system of any one of claims 1 to 24, wherein the dCasX comprises a sequence selected from the group consisting of SEQ ID NOs: 4 to 29, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. The system of claim 25, wherein the dCasX comprises the sequence of SEQ ID NO: 4, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. The system of claim 26, wherein the dCasX consists of the sequence of SEQ ID NO: 4. The system of any one of claims 1 to 27, wherein the RD1 comprises a sequence selected from the group consisting of SEQ ID NOs: 128 to 1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity therewith. The system of any one of claims 1 to 27, wherein the RD1 comprises a sequence selected from the group consisting of SEQ ID NOs: 130 to 138, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 130, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 131, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 132, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 133, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 134, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 135, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 136, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 137, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 138, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 7 to 15, 18 or 19 to 38, wherein the ADD comprises a sequence of SEQ ID NO: 125, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 39, wherein the DNMT3A comprises the sequence of SEQ ID NO: 126, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 40, wherein the DNMT3L comprises the sequence of SEQ ID NO: 127, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the dCasX comprises a sequence selected from the group consisting of SEQ ID NOs: 3122 and 22727, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NOs: 3334 to 6527, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NOs: 3334 to 3342 and 4931 to 4939, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. The system of any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3335 and SEQ ID NO: 4932, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3339 and SEQ ID NO: 4936, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3334 and SEQ ID NO: 4931, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3336 and SEQ ID NO: 4933, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3337 and SEQ ID NO: 4934, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3338 and SEQ ID NO: 4935, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3340 and SEQ ID NO: 4937, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3341 and SEQ ID NO: 4938, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3342 and SEQ ID NO: 4939, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 53, wherein the sequence encoding the DNMT3A comprises a sequence selected from the group consisting of SEQ ID NO: 3128 and SEQ ID NO: 3331, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 54, wherein the sequence encoding the DNMT3L comprises a sequence selected from the group consisting of SEQ ID NO: 3119 and SEQ ID NO: 3332, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 7 to 15, 18, 19 or 21 to 55, wherein the sequence encoding the ADD comprises a sequence selected from the group consisting of SEQ ID NOs: 3127, 3296 and 22726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOs: 6529 to 8134 and 14628 to 16233, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOs: 6529 to 6537 and 14628 to 14636, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6529 and SEQ ID NO: 14628, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6530 and SEQ ID NO: 14629, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6531 and SEQ ID NO: 14630, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6532 and SEQ ID NO: 14631, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6533 and SEQ ID NO: 14632, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6535 and SEQ ID NO: 14632, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6534 and SEQ ID NO: 14633, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6536 and SEQ ID NO: 14635, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6537 and SEQ ID NO: 4636, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOs: 9742 to 11347 and 17840 to 19446, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOs: 9742 to 9750 and 17841 to 17849, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9742 and SEQ ID NO: 17841, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9743 and SEQ ID NO: 17842, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9744 and SEQ ID NO: 17843, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9745 and SEQ ID NO: 17844, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9746 and SEQ ID NO: 17845, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9747 and SEQ ID NO: 17846, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9748 and SEQ ID NO: 17847, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9749 and SEQ ID NO: 17848, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9750 and SEQ ID NO: 17849, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 3, 8 to 15, 20 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOs: 8135 to 9740 and 16234 to 17839, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 3, 8 to 15, 20 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOs: 8135 to 8143 and 16234 to 16242, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 3, 7 to 15, 21 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOs: 11348 to 12953 and 19447 to 21052, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 3, 7 to 15, 21 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOs: 11348 to 11356 and 19447 to 19455, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 3 to 5, 7 to 15, 22, 23 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOs: 12954 to 14553 and 21053 to 22652, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1, 3 to 4, 6 to 15, 22 or 24 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOs: 14554 to 14626 and 22653 to 22725, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 84, wherein the mRNA is codon-optimized. A system as claimed in claim 85, wherein the mRNA is codon-optimized for expression in human cells. A system as claimed in any one of claims 1 to 86, wherein the mRNA is chemically modified, optionally wherein one or more or all uridine residues in the mRNA are replaced by 1-methyl-pseudouridine. The system of any one of claims 1 to 87, wherein the mRNA comprises a 5' untranslated region (UTR), a 3' UTR, a poly(A) sequence, and/or a 5' cap. The system of claim 88, wherein the 5'UTR comprises the sequence of SEQ ID NO: 3300. The system of claim 88 or claim 89, wherein the 3'UTR comprises the sequence of SEQ ID NO: 3310. A system as claimed in any one of claims 88 to 90, wherein the polyadenylation comprises the sequence of SEQ ID NO: 3057. A system as claimed in any one of claims 88 to 91, wherein the 5' cap is linked to the 5' of the mRNA. The system of any one of claims 87 to 92, wherein the mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 14628 to 16233, 16234 to 17839, 17841 to 19446, 19447 to 21052, 21053 to 22652, and 22653 to 22725, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. The system of any one of claims 87 to 92, wherein the mRNA comprises a sequence of SEQ ID NO: 14628 to 14635 or 14636, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. A system as in any one of claims 87 to 92, wherein the mRNA comprises the sequence of SEQ ID NO: 14628, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. A system as in any one of claims 87 to 92, wherein the mRNA comprises the sequence of SEQ ID NO: 14629, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. A system as in any one of claims 87 to 92, wherein the mRNA comprises the sequence of SEQ ID NO: 14630, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. A system as in any one of claims 87 to 92, wherein the mRNA comprises the sequence of SEQ ID NO: 14631, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. A system as in any one of claims 87 to 92, wherein the mRNA comprises the sequence SEQ ID NO: 14632, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. A system as in any one of claims 87 to 92, wherein the mRNA comprises the sequence of SEQ ID NO: 14633, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. A system as in any one of claims 87 to 92, wherein the mRNA comprises the sequence of SEQ ID NO: 14634, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. A system as in any one of claims 87 to 92, wherein the mRNA comprises the sequence of SEQ ID NO: 14635, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. A system as in any one of claims 87 to 92, wherein the mRNA comprises the sequence of SEQ ID NO: 14636, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. The system of any one of claims 1 to 103, wherein the PCSK9 gene target nucleic acid sequence is within 1.5 kb of the transcription start site (TSS) of the gene. The system of any one of claims 1 to 103, wherein the PCSK9 gene target nucleic acid sequence is within 500 bp upstream to 500 bp downstream of the TSS of the gene. The system of any one of claims 1 to 103, wherein the PCSK9 gene target nucleic acid sequence is within 300 bp upstream to 300 bp downstream of the TSS of the gene, or within 100 bp upstream to 100 bp downstream of the TSS of the gene. The system of any one of claims 1 to 103, wherein the PCSK9 gene target nucleic acid sequence is within 100 bp upstream to 100 bp downstream of the TSS of the gene. The system of any one of claims 1 to 107, wherein the PCSK9 gene target nucleic acid sequence is within 1 kb of an enhancer of the gene. The system of any one of claims 1 to 107, wherein the PCSK9 gene target nucleic acid sequence is within the 3' non-translated region of the PCSK9 gene. The system of any one of claims 1 to 109, wherein the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1824 to 1881, 1883 to 1897, 1899 to 1934, 1937 to 1991, 1993 to 1999, 2001 to 2026, 2029 to 2057, 2059 to 2106, 2108 to 2114, 2116 to 2122, 2124 to 2139, 2141 to 2209, 2211 to 2212, 2214 to 2233, 2235 to 2243 2252, 2254 to 2269, 2271 to 2281, 2283 to 2284, 2286 to 2320, 2322 to 2334, 2236 to 2346, 2348 to 2362, 2364 to 2375, 2377 to 2395, 2397 to 2428, 2430 to 2438, 2440 to 2463, 2465 to 2509, 2511 to 2544, 2672, 2675, 2694 and 2714. A system as claimed in any one of claims 1 to 109, wherein the targeting sequence of the gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 1824 to 1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694 and 2714. A system as claimed in any one of claims 1 to 109, wherein the targeting sequence of the gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 1834, 1849, 1853, 1855 to 1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874 and 1875. A system as claimed in any one of claims 1 to 109, wherein the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NO: 1855, 1867 and 1869. A system as claimed in any one of claims 110 to 113, wherein the targeting sequence of the gRNA comprises a sequence in which 1, 2, 3, 4 or 5 nucleotides are removed from the 3' end of the sequence. A system as claimed in any one of claims 1 to 114, wherein the gRNA is a single-molecule gRNA (sgRNA). A system as claimed in any one of claims 1 to 115, wherein the gRNA comprises a scaffold stem loop comprising a sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1822) or a sequence thereof having 1, 2, 3, 4 or 5 mismatches. The system of any one of claims 1 to 116, wherein the gRNA comprises a scaffold comprising a sequence selected from the group consisting of SEQ ID NOs: 1744 to 1746, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 116, wherein the gRNA comprises a scaffold comprising the sequence of SEQ ID NO: 1746, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto. A system as claimed in any one of claims 1 to 118, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 3074 to 3081, 3145 to 3147, 3150 to 3153 and 3158 to 3176, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith. A system as claimed in any one of claims 1 to 119, wherein the gRNA is chemically modified. A system as claimed in claim 120, wherein the chemical modification of the gRNA comprises adding a 2'O-methyl group to one or more nucleotides of the gRNA. A system as claimed in claim 121, wherein one or more of the 1, 2, 3 or 4 nucleotides at the 5' end, 3' end or both ends of the gRNA are modified by adding a 2'O-methyl group. A system as in any one of claims 120 to 122, wherein the chemical modification of the gRNA comprises substitution of a phosphorothioate bond between two or more nucleotides of the gRNA. The system of claim 123, wherein the chemical modification comprises substitution of a phosphorothioate bond between two or more nucleotides located at 1, 2, 3 or 4 nucleotides at the 5' end, 3' end or both ends of the gRNA. A system as claimed in any one of claims 104 to 124, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 3074 to 3081, 3147, 3151, 3153, 3159 to 3164, 3166 to 3171, 3173 to 3176 and 22788 to 22803, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith. A system as in any one of claims 104 to 124, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NO: 22788 to 22803. A system as in any one of claims 104 to 124, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NO: 22788 to 22790. A lipid nanoparticle comprising a system as described in any one of claims 1 to 127. A lipid nanoparticle (LNP) comprising an mRNA as described in any one of claims 87 to 103 and a gRNA as described in any one of claims 120 to 127. The LNP of claim 128 or 129, wherein the LNP comprises one or more components selected from the group consisting of: ionizable lipids, one or more auxiliary phospholipids, one or more lipids modified with polyethylene glycol (PEG), and cholesterol or its derivatives. The LNP of any one of claims 128 to 130, wherein the LNP comprises an ionizable lipid, a co-phospholipid, a lipid modified with polyethylene glycol (PEG), and cholesterol or a derivative thereof. The LNP of any one of claims 128 to 131, comprising a cationic lipid having a pKa of 5 to 8. A pharmaceutical composition comprising: a.   The system of any one of claims 1 to 127; or b. The LNP of any one of claims 128 to 132, and one or more pharmaceutically suitable excipients. A pharmaceutical composition comprising a plurality of lipid nanoparticles according to any one of claims 128 to 132, and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition of claim 134, wherein the average diameter of the plurality of lipid nanoparticles is between about 20 nm and about 200 nm. The pharmaceutical composition of claim 134 or claim 135, wherein the pharmaceutical composition is formulated for administration selected from the group consisting of intravenous, intraarterial, intraportal injection, intraperitoneal, intramuscular, intraventricular, intracisternal, intrathecal, intracranial, intralumbar, intraocular, subcutaneous and oral routes. A method for inhibiting the transcription of the PCSK9 gene in a cell population, the method comprising introducing into the cells of the population: a. a system as described in any one of claims 1 to 127; b. an LNP as described in any one of claims 128 to 132; c. a pharmaceutical composition as described in any one of claims 133 to 136; or d. a combination of two or more of (a) to (c), thereby inhibiting the transcription of the PCSK9 gene in the cell population. The method of claim 137, wherein transcription of the PCSK9 gene is inhibited in at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more of the cells in the population. The method of claim 137 or claim 138, wherein transcription of the PCSK9 gene in the cells of the population is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to untreated cells. A method as claimed in any one of claims 137 to 139, wherein the cells of the population are eukaryotic cells. The method of claim 140, wherein the eukaryotic cells are selected from the group consisting of rodent cells, mouse cells, rat cells, and non-human primate cells. The method of claim 140, wherein the eukaryotic cells are human cells. The method of any one of claims 140 to 142, wherein the eukaryotic cells are selected from the group consisting of hepatocytes, intestinal cells, kidney cells, central nervous system cells, smooth muscle cells, macrophages, and arterial wall cells. The method of any one of claims 137 to 143, wherein the inhibition of the PCSK9 gene in the cell population occurs in vitro or ex vivo. The method of any one of claims 137 to 143, wherein the inhibition of the PCSK9 gene in the cell population occurs in vivo in an individual. The method of claim 145, wherein the individual is selected from the group consisting of rodents, mice, rats, and non-human primates. The method of claim 145, wherein the individual is a human. A method as claimed in any one of claims 137 to 147, wherein the transcriptional inhibition is stabilized through one or more cell divisions. A method as claimed in any one of claims 137 to 148, wherein the transcriptional inhibition is heritable. A method as claimed in any one of claims 137 to 149, wherein the inhibition is reversible. The method of claim 150, wherein the inhibition can be reversed by using a DNMT inhibitor. The method of claim 151, wherein the DNMT inhibitor is a cytidine analog. The method of claim 151 or claim 152, wherein the DNMT inhibitor is selected from the group consisting of azacytidine, decitabine, clofarabine, and zebularine. A method for treating a PCSK9- related disease in an individual in need thereof, comprising administering a therapeutically effective amount of: a. a system as described in any one of claims 1 to 127; b. an LNP as described in any one of claims 128 to 132; or c. a pharmaceutical composition as described in any one of claims 133 to 136, thereby treating the PCSK9-related disease. The method of claim 154, wherein the subject is treated with a therapeutically effective amount of the LNP. The method of claim 154 or claim 155, wherein the LNP is administered by a route selected from the group consisting of intravenous, intraarterial, intraportal injection, intraperitoneal, intramuscular, intraventricular, intracisternal, intrathecal, intracranial, intralumbar, intraocular, subcutaneous, and oral routes. A method as in any one of claims 154 to 156, wherein the PCSK9- related disease is selected from the group consisting of: autosomal dominant hypercholesterolemia (ADH), hypercholesterolemia, elevated total cholesterol levels, hyperlipidemia, elevated low-density lipoprotein (LDL) levels, elevated LDL-cholesterol levels, decreased high-density lipoprotein levels, hepatic steatosis, coronary heart disease, ischemia, stroke, peripheral vascular disease, thrombosis, type 2 diabetes, hypertension, atherosclerosis, obesity, aortic stenosis, elevated PCSK9 levels, or a combination thereof. The method of any of claims 154 to 157, wherein the method results in improvement in at least one clinically relevant endpoint selected from the group consisting of: change from baseline in LDL-cholesterol, reduction in plaque atheroma volume, reduction in coronary plaque, reduction in atherosclerotic cardiovascular disease (ASCVD), cardiovascular death, non-fatal myocardial infarction, ischemic stroke, non-fatal stroke, coronary revascularization, unstable angina, and vision. The method of any of claims 154 to 158, wherein the method results in improvement in at least two clinically relevant endpoints selected from the group consisting of: change from baseline in LDL-cholesterol, reduction in plaque atheroma volume, reduction in coronary plaque, reduction in atherosclerotic cardiovascular disease (ASCVD), cardiovascular death, non-fatal myocardial infarction, ischemic stroke, non-fatal stroke, coronary revascularization, unstable angina, and vision. A kit comprising a system as in any one of claims 1 to 127, an LNP as in any one of claims 128 to 132, a pharmaceutical composition as in any one of claims 133 to 136, or a combination thereof, and a suitable container. The kit of claim 160, comprising a buffer, a filler, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label-displaying reagent, instructions for use, or any combination thereof. A composition comprising: a.   The system of any one of claims 1 to 127; b.   The LNP of any one of claims 128 to 132; or c.   The pharmaceutical composition of any one of claims 133 to 136. The composition of claim 162 is used for the manufacture of a medicament for treating a PCSK9- related disease in an individual in need thereof. The composition of claim 162, which is used to treat a PCSK9- related disease in an individual in need thereof.