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WO2022247873A1 - 工程化的Cas12i核酸酶、效应蛋白及其用途 - Google Patents

工程化的Cas12i核酸酶、效应蛋白及其用途 Download PDF

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WO2022247873A1
WO2022247873A1 PCT/CN2022/095072 CN2022095072W WO2022247873A1 WO 2022247873 A1 WO2022247873 A1 WO 2022247873A1 CN 2022095072 W CN2022095072 W CN 2022095072W WO 2022247873 A1 WO2022247873 A1 WO 2022247873A1
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cas12i
nuclease
engineered
amino acid
amino acids
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PCT/CN2022/095072
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English (en)
French (fr)
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李伟
周琪
陈阳灿
胡艳萍
王鑫阁
骆胜球
陈逸
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中国科学院动物研究所
北京干细胞与再生医学研究院
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Priority claimed from CN202110581290.3A external-priority patent/CN113151215B/zh
Application filed by 中国科学院动物研究所, 北京干细胞与再生医学研究院 filed Critical 中国科学院动物研究所
Priority to CN202280035920.XA priority Critical patent/CN117337326A/zh
Priority to JP2023573318A priority patent/JP2024522112A/ja
Priority to EP22810597.9A priority patent/EP4349979A4/en
Publication of WO2022247873A1 publication Critical patent/WO2022247873A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • Genome editing is an important and useful technique in genome research.
  • Several systems are available for genome editing, including clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems, transcription activator-like effector nuclease (TALEN) systems, and zinc finger nuclease (ZFN) systems.
  • CRISPR clustered regularly interspaced short palindromic repeat
  • TALEN transcription activator-like effector nuclease
  • ZFN zinc finger nuclease
  • type II Cas9 system and type V-A/B/E/J Cas12a/Cas12b/Cas12e/Cas12j system have been exploited for genome editing and provide a broad field for biomedical research. prospect.
  • Cas12i nuclease of a kind of engineering It comprises one or more mutations based on reference Cas12i nuclease, said mutation is selected from:
  • the one or more amino acids that interact with PAM are one or more of the following amino acids: E176, K238, T447 and E563,
  • amino acid position numbers are as defined in the corresponding amino acid positions shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease as described in any one of items 1-3, wherein, the replacement of one or more amino acids interacting with PAM in the reference Cas12i nuclease with a positively charged amino acid is Refers to one or more of the following substitutions: E176R, K238R, T447R and E563R;
  • the one or more amino acids involved in opening the DNA double strand are one or more amino acids at the following positions: 163 and 164;
  • amino acid position numbers are as defined in the corresponding amino acid positions shown in SEQ ID NO:1.
  • the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are one or more amino acids at the following positions: 323, 327, 355, 359, 360, 361, 362, 388, 390, 391, 392, 393, 414, 417, 418, 421, 424, 425, 650, 652, 653, 696, 705, 708, 709, 751, 752, 755, 840, 848, 851, 856, 885, 897, 925, 926, 928, 929, 932, 1022.
  • the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are one or more of the following amino acids: E323, L327, V355, G359, G360, K361, D362, L388, N390 , N391, F392, K393, Q414, L417, L418, K421, Q424, Q425, S650, E652, G653, I696, K705, K708, E709, L751, S752, E755, N840, N848, S851, A856, Q885, M897 , N925, I926, T928, G929, Y932, A1022;
  • the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are one or more of the following amino acids: E323, D362, Q425, N925, I926, N391, Q424 and G929;
  • amino acid position numbers are as defined in the corresponding amino acid positions shown in SEQ ID NO:1.
  • the positively charged amino acid includes substitution by R or K, preferably, the positively charged amino acid is R.
  • the engineered Cas12i nuclease comprises any of the following mutations or mutation combinations: (1) E323R; (2) D362R; (3) Q425R; (4) N925R; (5) I926R; (6) E323R (7) E323R and Q425R; (8) E323R and I926R; (9) Q425R and I926R; (10) D362R and I926R; (11) N925R and I926R; (12) E323R, D362R and Q425R; (13) E323R, D362R and I926R; (14) E323R, Q425R and I926R; (15) D362R, N925R and I926R; and (16) E323R, D362R, Q425R and I926R;
  • amino acid position is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • the one or more amino acids that interact with the DNA-RNA double helix are one or more of the following amino acids: G116, E117, A156, T159, S161, T301, I305, K306, T308, N312, F313, D427, K433, V438, N441, Q442, M852, L855, N861, Q865, E160, Q316, E319, Q320, E247, E343, E348, E349, N679, E683, E691, D782, E783, E797, E800, D853, S957, D958, G293, E294 and N297; G116, E117, A156, T159, E160, S161, E247, G293, E294, N297, 1301, 1305, K306, T308, N312, F313, Q316, E319, Q320, E343, E348, E349, D427, K433, V438, N441, Q442, N679, E683, E
  • amino acid position is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • one or more polar or positively charged amino acids interacting with the DNA-RNA double helix are selected from one or more of the following Amino acids at multiple positions: 357, 394, 715, 719, 807, 844, 848, 857, 861, that is, one or more of the following amino acids: H357, K394, R715, R719, K807, K844, N848, R857, R861 ;
  • amino acid position is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • amino acid position is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • the flexible region mutation is located at position 439 and/or 926;
  • the flexible region mutation is a mutation of L439 and/or I926;
  • amino acid position is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • said one or more flexible region mutations comprise I926G, L439(L+G) or L439(L+GG);
  • the one or more flexible region mutations comprise L439(L+G) or L439(L+GG);
  • amino acid position numbers are as defined in the corresponding amino acid positions shown in SEQ ID NO:1.
  • the Cas12i nuclease of described engineering comprises any following one or more sets of mutations:
  • amino acid position is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • An engineered Cas12i nuclease comprising any of the following mutations: (1) E176R, K238R, T447R, E563R and N164Y; (2) E176R, K238R, T447R, E563R and I926R; (3) N164Y, E323R and D362R; (4) E176R, K238R, T447R, E563R, E323R and D362R; (5) N164Y and I926R; (6) E176R, K238R, T447R, E563R, N164Y and I926R; (7) )E176R, K238R, T447R, E563R, N164Y, E323R, and D362R; (8) E176R, K238R, T447R, E563R, N164Y, I926R, E323R, and D362R; (9) E176R, K238R, T447R, E56
  • An engineered Cas12i effector protein comprising the engineered Cas12i nuclease or a functional derivative thereof according to any one of items 1 to 22;
  • the engineered Cas12i nuclease or its functional derivative has enzymatic activity, or the engineered Cas12i nuclease or its functional derivative is an enzyme inactive mutant.
  • engineered Cas12i effector protein as described in item 23, wherein said engineered Cas12i nuclease or its functional derivative is an enzyme inactivation mutant comprising one or more of the following mutations: D599A, E833A, S883A , H884A, R900A and D1019A; wherein, said amino acid position is as defined in the corresponding amino acid position shown in SEQ ID NO: 1.
  • the engineered Cas12i effector protein according to any one of items 23 to 25, further comprising a functional domain fused with the engineered Cas12i nuclease or a functional derivative thereof.
  • the engineered Cas12i effector protein according to any one of items 23-27, the engineered Cas12i effector protein comprising: the N-terminal part containing the engineered Cas12i nuclease or its functional derivative A first polypeptide and a second polypeptide comprising the C-terminal portion of the engineered Cas12i nuclease or a functional derivative thereof, wherein the first polypeptide and the second polypeptide are capable of following a guide comprising a guide sequence associate with each other in the presence of the RNAs to form clustered regularly interspaced short palindromic repeat (CRISPR) complexes that specifically bind to a target nucleic acid comprising a target sequence complementary to the guide sequence;
  • CRISPR clustered regularly interspaced short palindromic repeat
  • the first polypeptide comprises amino acid residues 1 to X of the N-terminal portion of the engineered Cas12i nuclease described in any one of items 1-22, and the second polypeptide comprises any of items 1-22 An amino acid residue X+1 of the engineered Cas12i nuclease to the C-terminus of the Cas12i nuclease;
  • said first polypeptide and said second polypeptide each comprise a dimerization domain
  • said first dimerization domain and said second dimerization domain associate with each other in the presence of an inducing agent.
  • An engineered CRISPR-Cas12i system comprising:
  • a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding said guide RNA.
  • the engineered Cas12i nuclease or the engineered Cas12i effector protein and the guide RNA can form a CRISPR complex, and the CRISPR complex specifically binds to a target nucleic acid comprising the target sequence and induces the target nucleic acid modification;
  • the guide RNA is a crRNA comprising the guide sequence
  • the engineered CRISPR-Cas12i system includes a precursor guide RNA array (array) encoding multiple crRNAs;
  • the engineered Cas12i nuclease or the engineered Cas12i effector protein is a master editor
  • the guide RNA is a guide editing guide RNA (pegRNA).
  • said one or more vectors are selected from the group consisting of retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors and herpes simplex vectors;
  • the one or more vectors are adeno-associated virus AAV vectors
  • the AAV vector also encodes the guide RNA.
  • a method for detecting target nucleic acid in a sample comprising:
  • a method of modifying a target nucleic acid comprising a target sequence comprising contacting said target nucleic acid with the engineered CRISPR-Cas12i system described in item 29 or 30;
  • the method is performed in vitro, ex vivo or in vivo;
  • the target nucleic acid is present in a cell
  • the cells are bacterial cells, yeast cells, mammalian cells, plant cells or animal cells;
  • the target nucleic acid is genomic DNA
  • the engineered CRISPR-Cas12i system comprises a precursor guide RNA array encoding a plurality of crRNAs, wherein each crRNA comprises a different guide sequence.
  • the engineered CRISPR-Cas12i system as described in item 29 or 30 in the preparation of a drug for treating a disease or a disease associated with a target nucleic acid in an individual's cells; preferably, the disease or disease is selected from the group consisting of: Cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection and viral infection.
  • a method of treating a disease or disorder associated with a target nucleic acid in a cell of an individual comprising using the method of item 32 to modify the target nucleic acid in the cell of the individual, thereby treating the disease or A disorder; preferably, said disease or disorder is selected from the group consisting of cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection and viral infection.
  • a method of modifying a target nucleic acid comprising a target sequence comprising contacting said target nucleic acid with the engineered CRISPR-Cas12i system described in item 29 or 30.
  • An engineered non-human animal comprising one or more engineered cells according to item 37.
  • the Cas12i nuclease engineered in this application and its effector protein have higher activity, such as the catalytic efficiency of cutting nucleic acid substrates and the gene editing efficiency in cells.
  • the engineered Cas12i nuclease in the present application has more excellent gene editing efficiency in mammalian cells (such as human cells) than existing conventional Cas gene editing tools;
  • the gene editing efficiency of multiple sites (such as 62 sites) in the cell was tested, and it was found that the gene editing efficiency of 57 sites exceeded about 60%, and the average gene editing efficiency was close to 70%.
  • the engineered Cas12i nuclease and its effector protein of the present application also have one or more of the following advantages: the protein is small (1,054aa), the crRNA component is simple, the PAM sequence is simple, and the protein itself can process the precursor crRNA.
  • the application provides Cas12i nuclease with lower off-target rate and higher specificity further artificially modified on the basis of highly active engineered Cas12i nuclease (such as SEQ ID NO.8).
  • Figure 1a Amino acids that interact with PAM in the reference Cas12i nuclease (wild-type Cas12i nuclease shown in SEQ ID NO.1) were replaced with positively charged amino acids to improve gene editing efficiency. As shown in the figure, the four mutants E176R, K238R, T447R, and E563R can significantly improve the gene editing efficiency in human 293T cells.
  • Figure 1b Combining the amino acid mutations (E176R, K238R, T447R, E563R) that can significantly improve gene editing efficiency in Figure 1a, it was found that the combined mutants can exhibit higher gene editing efficiency in human 293T cells.
  • Figure 2 The amino acid involved in opening the DNA double strand in the reference Cas12i nuclease is replaced with an amino acid with an aromatic ring to improve the efficiency of gene editing.
  • mutants such as Q163F, Q163Y, Q163W, N164F, and N164Y can significantly improve the gene editing efficiency in human 293T cells.
  • Figure 3a, 3b, 3c Amino acids in the reference Cas12i nuclease located in the RuvC domain and interacting with single-stranded DNA substrates were replaced with positively charged amino acids, thereby improving the efficiency of gene editing.
  • mutants such as E323R, L327R, V355R, G359R, G360R, D362R, N391R, Q424R, Q425R, N925R, I926R, and G929R can significantly improve the gene editing efficiency in human 293T cells.
  • Figure 3d Combining the efficiency-enhancing point mutations in Figures 3a and 3b, it was found that the combined mutants can exhibit higher gene editing efficiency in human 293T cells.
  • Figure 3e Combining the efficiency-enhancing point mutations in Figures 3a and 3b and the modified mutations (L439(L+GG), I926G) based on the principle of molecular flexibility, it was found that the combined mutants can exhibit higher efficiencies in human 293T cells Gene editing efficiency.
  • Figure 4 Replacing amino acids that interact with the DNA-RNA duplex in the reference Cas12i enzyme with positively charged amino acids improves gene editing efficiency.
  • the G116R, E117R, T159R, S161R, E319R, E343R, and D958R mutants can significantly improve the gene editing efficiency in human 293T cells.
  • the D958R mutation is the best.
  • FIG 5 Combining the high-efficiency mutants obtained from the three transformation strategies in Figure 1a to Figure 3e and the transformation mutations (L439(L+GG), I926G) based on the principle of molecular flexibility, it is found that the combined mutants can display Higher gene editing efficiency in 293T cells. The combination can greatly improve the efficiency of gene editing.
  • the mutant with the best gene editing effect was selected and named CasXX for subsequent experiments.
  • CasXX (SEQ ID NO:8) is a Cas12i engineered enzyme with E176R+K238R+T447R+E563R+N164Y+E323R+D362R mutation combination (reference Cas12i2 based on amino acid sequence SEQ ID NO:1).
  • Figure 6a Summary of gene editing efficiencies of CasXX at 62 human genomic loci.
  • PAM NTTN.
  • Figure 6b Comparison of gene editing efficiency between CasXX and AsCas12a, BhCas12b v4.
  • Figure 6c Comparison of gene editing efficiency between CasXX and SpCas9, SaCas9, SaCas9-KKH.
  • Figure 6d Statistics of gene editing efficiency of CasXX in the mouse Hepa1-6 cell line. It can be seen that CasXX exhibited powerful gene editing capabilities at 65 sites in mouse Hepa1-6 cells, with an average gene editing efficiency of more than 60%.
  • Figure 7 shows the gene editing efficiency of CasXX at 64 human loci.
  • the PAM sequences contained in these 64 sites cover all NNNN combinations: NTTN, NTAN, NTCN, NTGN, NATN, NAAN, NACN, NAGN, NCTN, NCAN, NCCN, NCGN, NGTN, NGAN, NGCN, NGGN.
  • CasXX exhibited high gene editing efficiency in PAM sequences such as NTTN, NTAN, NTCN, NTGN, NATN, NAAN, NCTN, NCAN, and NGTN, and the average gene editing efficiency at these sites exceeded 40%.
  • Figure 8 shows the results of wild-type Cas12i2 protein (SEQ ID NO: 1) and CasXX protein cutting double-stranded DNA in vitro.
  • the wild-type Cas12i2 protein only has partial cutting efficiency for double-stranded DNA containing NTTN PAM, but has almost no cutting activity for the rest of the PAM.
  • CasXX protein showed high cutting efficiency for double-stranded DNA containing NTTN, NTAN, NTCN, NTGN, NATN, NAAN, NACN, NCTN, NCAN, NGTN, NGAN PAM.
  • Figure 9 Homology alignment of the amino acid sequences of Cas12i2 (SEQ ID NO: 1) and Cas12i1 (SEQ ID NO: 13).
  • the shaded amino acids represent the same amino acids in the two Cas12i proteins, and the amino acids marked in white boxes represent the amino acids in the two Cas12i proteins with similar properties.
  • Figure 10 is the use of GUIDE-Seq to detect the off-target effect of CasXX at the EMX1-7 target site.
  • the numbers after the sequence represent the number of reads (reads) detected for each sequence.
  • the first line of sequence is the reference target sequence (SEQ ID NO: 77), and the following sequences represent the target sequence and off-target sequence and the reads (reads) enriched by double-stranded DNA tags in the target sequence and off-target sequence, respectively.
  • SEQ ID NO: 77 the reference target sequence
  • This figure shows that CasXX has off-target effects at the EMX1-7 site.
  • Figure 11 shows the construction of new HF mutants by adding new point mutations or deleting original point mutations on the basis of the original sequence of CasXX.
  • the experimental results showed that R857A, N861A, K807A, N848A, R715A, R719A, K394A, H357A, K844A, these single point mutants based on CasXX sequence could effectively reduce the EMX1-7-OT-1, EMX1-7-OT-2 , indel ratio of emx1-7-ot-3.
  • Figure 12 shows the construction of new HF mutants by adding new point mutations or deleting original point mutations on the basis of the original sequence of CasXX.
  • the experimental results showed that R857A, N861A, K807A, N848A, R715A, R719A, K394A, H357A, K844A, these single point mutants based on CasXX sequence were used to mimic off-target crRNA-RNF2-1-Mis-1/2, crRNA-RNF2 -1-Mis-5/6, crRNA-RNF2-1-Mis-17/18 and crRNA-RNF2-1-Mis-19/20 can effectively reduce the ratio of indels.
  • FIG. 13 is an experimental process for screening mutants (CasXX-based HF mutants) that improve the specificity of gene editing using a fluorescent reporter system. 600ng of a plasmid encoding Cas protein, 300ng of a plasmid encoding crRNA and 100ng of a plasmid encoding mCherry were transfected into cells cultured in a 24-well cell culture dish. Three days after transfection, the proportion of remaining mCherry positivity for each sample was calculated using flow cytometry. The calculation formula is shown in the figure.
  • Figure 14 shows that CasXX and mutants edit the mCherry gene located on the plasmid in 293T cells. After plasmid transfection, cultured for three days, flow cytometric analysis was performed. The higher the gene editing efficiency of the Cas protein, the lower the proportion of mCherry-positive cells.
  • the experimental process corresponds to the schematic diagram of the experimental process in Figure 13; the spacer sequences for mCherry-FM, mCherry-Mis-1/2, mCherry-Mis-5/6 and mCherry-Mis-19/20 are respectively as SEQ ID NO: 37 , 38, 39 and 40.
  • Figure 15 shows the construction of a new combination of HF mutants based on the original sequence of CasXX.
  • the experimental results showed that CasXX sequence-based mutants R857A, R719A, K394A, K844A, R719A/K844A and R857A/K844A could effectively reduce the EMX1-7-OT-1, EMX1-7-OT-2, EMX1-7 - OT-3 off-target indel ratio without sacrificing efficiency at on-target sites.
  • Figure 16 shows the construction of a new combination of HF mutants based on the original sequence of CasXX.
  • the experimental results showed that R857A, R719A, K394A, K844A, R719A/K844A and R857A/K844A mutants based on the CasXX sequence were used to mimic off-target crRNA-RNF2-1-Mis-1/2, crRNA-RNF2-1-Mis -5/6, crRNA-RNF2-1-Mis-17/18 and crRNA-RNF2-1-Mis-19/20 can effectively reduce the ratio of indels without sacrificing the efficiency at the target site.
  • Figure 17 is the use of GUIDE-Seq to detect the off-target effect of CasXX and CasXX+K394A mutant at the CD34-7 target site.
  • the number after the sequence represents the number of reads detected for each sequence.
  • the first line of sequence is the reference target sequence (SEQ ID NO: 78), and the following sequences respectively represent the target sequence and off-target sequence and the number of reads enriched by double-stranded DNA tags in the target sequence and off-target sequence.
  • SEQ ID NO: 78 the reference target sequence
  • This figure shows that CasXX has an off-target effect at the CD34-7 site, but the CasXX+K394A mutant has no off-target effect at this site.
  • effector protein refers to an activity such as site-specific binding activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, single-stranded RNA cleavage activity, DNA or RNA modification (such as cleavage, base substitution, insertion, removal) or transcriptional regulatory activity.
  • guide RNA and “gRNA” are used interchangeably herein to refer to an RNA capable of forming a complex with a Cas12i effector protein and a target nucleic acid (eg, double-stranded DNA). Also considered herein are arrays of precursor guide RNAs that can be processed into multiple crRNAs.
  • crRNA or “CRISPR RNA” comprises a guide sequence having sufficient complementarity to the target sequence of a target nucleic acid (e.g., double-stranded DNA) that directs the specific binding of the CRISPR complex to the target sequence of the target nucleic acid.
  • CRISPR array refers to a nucleic acid (e.g., DNA) segment comprising CRISPR repeats and spacers, starting at the first nucleotide of the first CRISPR repeat and ending at the end of the last (terminal) CRISPR repeat. A nucleotide ends. Typically, each spacer in a CRISPR array is located between two repeats.
  • CRISPR repeat or “CRISPR direct repeat” or “direct repeat” as used herein refers to a plurality of short direct repeat sequences that exhibit very little or no sequence variation in a CRISPR array. Suitably, direct repeats can form stem-loop structures.
  • nucleic acid refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analog.
  • Oligonucleotide are used interchangeably to refer to short polynucleotides having no more than about 50 nucleotides.
  • complementarity refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through conventional Watson-Crick base pairing.
  • Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., 5, 6, 7, 8, 9, 10, complementary about 50%, 60%, 70%, 80%, 90% and 100%, respectively).
  • Perfectly complementary means that all contiguous residues of a nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence.
  • substantially complementary refers to a degree of complementarity of at least about 70%, over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, Any of 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100, or refers to two nucleic acids that hybridize under stringent conditions.
  • stringent conditions for hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence primarily hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence will specifically hybridize to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nuclear acid probe assay," Elsevier, N, Y.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogstein bonding, or in any other sequence-specific manner. A sequence that is capable of hybridizing to a given sequence is called the "complement" of that given sequence.
  • Percent (%) sequence identity for a nucleic acid sequence is defined as the difference between a candidate sequence and a specific nucleic acid sequence after aligning the sequences (if necessary) by allowing gaps (gaps) to achieve the maximum percent sequence identity The percentage of nucleotides that are identical to each other.
  • Percent sequence identity (%) for a peptide, polypeptide or protein sequence is the number of sequences in a candidate sequence that are identical to a particular peptide or The percentage of amino acid residues that are substituted with identical amino acid residues in the amino acid sequence.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available programs such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • polypeptide and “peptide” are used interchangeably herein to refer to a polymer of amino acids of any length.
  • the polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids.
  • a protein can have one or more polypeptides.
  • the term also encompasses amino acid polymers that have been modified; eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as conjugation with a labeling component).
  • variant is interpreted as a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs from the nucleic acid sequence of another reference polynucleotide. Changes in the nucleic acid sequence of a variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as described below.
  • a typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Usually, the differences are limited such that the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions.
  • the amino acid sequence of a variant and reference polypeptide may differ by any combination of one or more substitutions, additions, deletions.
  • the substituted or inserted amino acid residues may or may not be those encoded by the genetic code.
  • Variants of a polynucleotide or polypeptide may be naturally occurring (such as allelic variants), or may be variants that are not known to occur naturally.
  • Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.
  • wild type has the meaning commonly understood by those skilled in the art, meaning a typical form of an organism, strain, strain, gene or trait. It can be isolated from resources in nature and has not been deliberately modified.
  • nucleic acid molecule or polypeptide when used to describe a nucleic acid molecule or polypeptide, mean that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated or occurs in nature.
  • an "ortholog" of a protein as referred to herein refers to a protein belonging to a different species that performs the same or a similar function as the protein that is its ortholog.
  • identity is used to denote a sequence match between two polypeptides or between two nucleic acids.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides one position of , is occupied by lysine)
  • every molecule is identical at that position.
  • the "percent identity" between these two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions of the two sequences match.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • comparisons are made when two sequences are aligned to yield maximum identity.
  • This alignment can be achieved, for example, by the method of Needleman et al. (1970) J.Mol.Biol. Inc.) to carry out.
  • the PAM 120 weight residue table can also be used, integrated into the ALIGN program (version 2.0) using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)).
  • a gap length penalty of 12 and a gap penalty of 4 are used to determine the percent identity between two amino acid sequences.
  • the algorithm of Needleman and Wunsch J MoI Biol.
  • Cell as used herein is understood not only to refer to a specific single cell, but also to the progeny or potential progeny of that cell. Because certain modifications may have occurred in the progeny, due to mutations or environmental influences, such progeny may in fact differ from the parental cells and still be included within the scope of the term herein.
  • transduction and “transfection” include methods known in the art to introduce DNA into cells to express a protein or molecule of interest using an infectious agent such as a virus or otherwise.
  • infectious agent such as a virus or otherwise.
  • chemical-based transfection methods such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers such as DEAE-dextran or polyethyleneimine.
  • transfection methods non-chemical methods such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, plasmid delivery, or transposons; particle-based Methods such as the use of gene guns, magnetofection or magnet-assisted transfection, particle bombardment; and hybridization methods such as nucleofection.
  • transfected refers to the process of transferring or introducing exogenous nucleic acid into a host cell.
  • a “transfected”, “transformed” or “transduced” cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid.
  • in vivo refers to the organism from which cells are obtained. "Ex vivo” or “in vitro” means outside the organism from which cells are obtained.
  • treatment/treating is a method used to obtain beneficial or desired results, including clinical results.
  • beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease, reduction of the extent of the disease, stabilization of the disease (e.g., prevention or delay of the disease) exacerbation of the disease), preventing or delaying the spread of the disease (e.g.
  • metastasis preventing or delaying the recurrence of the disease, reducing the rate of recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing remission (partial or total) of the disease, To reduce the dose of one or more other drugs needed to treat the disease, delay disease progression, improve quality of life, and/or prolong survival.
  • Treatment also includes reducing the pathological consequences of a disorder, condition or disease. The methods of the invention contemplate any one or more of these aspects of treatment.
  • the term "effective amount” refers to an amount of a compound or composition sufficient to treat a particular disorder, condition or disease, eg, ameliorate, alleviate, lessen and/or delay one or more symptoms thereof.
  • an "effective amount” may be administered in one or more doses, ie, a single dose or multiple doses may be required to achieve the desired therapeutic endpoint.
  • Subject “Subject”, “individual” or “patient” are used interchangeably herein for purposes of treatment and refer to any animal classified as a mammal, including humans, livestock and farm animals, and zoo, farm or Pet animals such as dogs, horses, cats, cows, etc.
  • the individual is a human individual.
  • references to "not” values or parameters generally mean and describe “except” values or parameters.
  • the method is not used to treat type X cancer, meaning that the method is used to treat cancer other than type X.
  • the term “and/or” in words such as “A and/or B” is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” in words such as "A, B and/or C” is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • an engineered Cas12i nuclease comprises one or more (for example two, three, four or five) of the following based on the reference Cas12i Nuclease mutations selected from the group consisting of:
  • the reference Cas12i nuclease is a natural Cas12i nuclease, such as the wild-type Cas12i2 nuclease whose amino acid sequence is as shown in SEQ ID NO: 1.
  • the reference Cas12i nuclease is a variant of the Cas12i nuclease, such as a natural variant.
  • the reference Cas12i nuclease is an engineered Cas12i (such as Cas12i2 or Cas12i1) nuclease that does not comprise one or more mutations in (1)-(5) above.
  • the amino acid position is defined as the corresponding amino acid position shown in SEQ ID NO: 1. In some embodiments, the amino acid position is as defined by the amino acid position corresponding to SEQ ID NO: 1 of a wild-type Cas12i nuclease having a sequence different from SEQ ID NO: 1.
  • the amino acid is at the X position, wherein said amino acid position is defined as the corresponding amino acid position of the wild-type Cas12i nuclease shown in SEQ ID NO: 1
  • the amino acid is at the X position, wherein said Said amino acid position is defined as the amino acid position of the corresponding SEQ ID NO: 1 of the wild-type Cas12i nuclease having a sequence different from SEQ ID NO: 1
  • the amino acid residue is located at a certain position of the reference enzyme Cas12i , which is quite at the X position of SEQ ID NO: 1, and the amino acid sequence of the reference enzyme Cas12i is aligned with the amino acid sequence of SEQ ID NO: 1 based on sequence homology.
  • Figure 7 shows the homology alignment of the amino acid sequences of CAS12i2 (SEQ ID NO: 1) and CAS12i1 (SEQ ID NO: 13).
  • CAS12i2 SEQ ID NO: 1
  • CAS12i1 SEQ ID NO: 13
  • Those skilled in the art can use software commonly used in the art, such as Clustal Omega, to compare and align (alignment) the amino acid sequence of any reference Cas12i nuclease with SEQ ID NO: 1, and then obtain the amino acid sequence consistent with the present application.
  • the amino acid position in the reference Cas12i nuclease corresponding to the amino acid position defined based on SEQ ID NO: 1.
  • the present application provides methods for engineering enzymes by introducing amino acid mutations based on any one or combination of more of the above five engineering principles, which lead to in vitro and/or in vivo enzyme activity (such as DNA single-strand or double-strand cleavage activity) (for example, the cutting gene efficiency increased by about 100 times), the increase in the number of recognizable PAMs (for example, as can be seen from Figure 8, the wild-type enzyme can recognize a PAM, and based on the above Amino acid mutated CasXX enzymes can recognize at least 11 PAMs), and/or reduce the off-target rate and improve specificity (for example, as can be seen from Table 18, the off-target efficiency of HF-Cas can be reduced by about 99% relative to CasXX).
  • in vitro and/or in vivo enzyme activity such as DNA single-strand or double-strand cleavage activity
  • the wild-type enzyme can recognize a PAM
  • Amino acid mutated CasXX enzymes can recognize at least 11 PAM
  • the engineered Cas12i nuclease contains one or more specific mutations as described in sections 1)-6) below. In some embodiments, any one or more mutations described in the application can be combined with existing Cas12i mutations (such as the mutations described in the following 7) section) to provide engineered Cas12i nucleic acids with higher activity enzyme.
  • an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with a positively charged amino acid mutation. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises replacing one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease with an aromatic ring amino acid.
  • an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises one or more elements that will be located in the RuvC domain in the reference Cas12i nuclease and interact with a single-stranded DNA substrate. Amino acids are replaced with positively charged amino acids.
  • an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises one or more amino acid amino acids that interact with the DNA-RNA double helix in the reference Cas12i nuclease are replaced by Positively charged amino acids.
  • an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises one or more polarities or bands that interact with the DNA-RNA duplex in the reference Cas12i nuclease. Positively charged amino acids are replaced with hydrophobic amino acids.
  • an engineered Cas12i nuclease comprises: 1) replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positive and 2) one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease are replaced by amino acids with an aromatic ring.
  • an engineered Cas12i nuclease comprises: 1) replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positive and 2) replacing one or more amino acids located in the RuvC domain in the reference Cas12i nuclease and interacting with single-stranded DNA substrates with positively charged amino acids.
  • an engineered Cas12i nuclease comprises: 1) one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease are replaced with a band Amino acids of the aromatic ring; and 2) one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate in the reference Cas12i nuclease are replaced with positively charged amino acids.
  • the engineered Cas12i nuclease further comprises replacing one or more polar or positively charged amino acids that interact with the DNA-RNA duplex in the reference Cas12i nuclease with hydrophobic amino acids.
  • the engineered Cas12i nuclease also comprises one or more mutations in the flexible region (such as replacing G, and/or inserting one or two Gs thereafter) to increase the flexibility of the flexible region.
  • the Cas12i engineered enzyme so modified has increased flexibility by at least about 10%, such as by at least about 20%, 30%, 50%, 100%, 150%, 200%, 500%, 1000% % or greater degree of flexibility.
  • an engineered Cas12i nuclease comprises: 1) replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positive and 2) replacing one or more amino acids that interact with the DNA-RNA duplex in the reference Cas12i nuclease with a positively charged amino acid.
  • an engineered Cas12i nuclease comprises: 1) one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease are replaced with a band Amino acids of the aromatic ring; and 2) replacing one or more amino acids that interact with the DNA-RNA double helix in the reference Cas12i nuclease with positively charged amino acids.
  • an engineered Cas12i nuclease comprises: 1) the RuvC domain and interacting with the single-stranded DNA substrate in the reference Cas12i nuclease One or more amino acids are replaced with positively charged amino acids; and 2) one or more amino acids that interact with the DNA-RNA double helix in the reference Cas12i nuclease are replaced with positively charged amino acids.
  • the engineered Cas12i nuclease further comprises replacing one or more polar or positively charged amino acids that interact with the DNA-RNA duplex in the reference Cas12i nuclease with hydrophobic amino acids.
  • the engineered Cas12i nuclease also comprises one or more mutations in the flexible region to increase the flexibility of the flexible region.
  • an engineered Cas12i nuclease comprises: 1) replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positive 2) replace one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease with an amino acid with an aromatic ring; and 3) replace the RuvC domain in the reference Cas12i nuclease with the One or more amino acids that interact with the single-stranded DNA substrate are replaced with positively charged amino acids.
  • an engineered Cas12i nuclease comprises: 1) replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positive 2) one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease are replaced by amino acids with an aromatic ring; and 3) the reference Cas12i nuclease is combined with the DNA-RNA double helix Interacting amino acid(s) are replaced with positively charged amino acids.
  • an engineered Cas12i nuclease comprises: 1) replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positive 2) replacing one or more amino acids located in the RuvC domain in the reference Cas12i nuclease and interacting with the single-stranded DNA substrate with a positively charged amino acid; and 3) replacing the reference Cas12i nucleic acid One or more amino acids in the enzyme that interact with the DNA-RNA double helix are replaced with positively charged amino acids.
  • an engineered Cas12i nuclease comprises: 1) one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease are replaced with a band 2) replace one or more amino acids in the reference Cas12i nuclease that are located in the RuvC domain and interact with the single-stranded DNA substrate with positively charged amino acids; and 3) replace the reference Cas12i nuclease One or more amino acids that interact with the DNA-RNA double helix are replaced with positively charged amino acids.
  • the engineered Cas12i nuclease further comprises replacing one or more polar or positively charged amino acids that interact with the DNA-RNA duplex in the reference Cas12i nuclease with hydrophobic amino acids. In some embodiments, the engineered Cas12i nuclease also comprises one or more mutations in the flexible region to increase the flexibility of the flexible region.
  • an engineered Cas12i nuclease comprises: 1) replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positive 2) one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease are replaced by amino acids with an aromatic ring; 3) the reference Cas12i nuclease is located in the RuvC domain and is combined with a single One or more amino acids that interact with the strand DNA substrate are replaced with positively charged amino acids; and 4) one or more amino acids that interact with the DNA-RNA double helix in the reference Cas12i nuclease are replaced with positively charged amino acids of amino acids.
  • the engineered Cas12i nuclease further comprises replacing one or more polar or positively charged amino acids that interact with the DNA-RNA duplex in the reference Cas12i nuclease with hydrophobic amino acids.
  • an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positive 2) one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease are replaced by amino acids with an aromatic ring; 3) the reference Cas12i nuclease is located in the RuvC domain and is combined with a single One or more amino acids interacting with the strand DNA substrate are replaced with positively charged amino acids; 4) one or more amino acids interacting with the DNA-RNA double helix in the reference Cas12i nuclease are replaced with positively charged amino acids Amino acids; and 5) replacing one or more polar
  • the engineered Cas12i nuclease comprises a mutation relative to the corresponding amino acid position shown in SEQ ID NO: 1: N164Y+E176R+K238R+E323R+D362R+T447R+E563R (henceforth named "CasXX ").
  • the Cas12i nuclease of described engineering comprises the sequence of SEQ ID NO:8.
  • the engineered Cas12i nuclease comprises one or more mutations based on a reference Cas12i nuclease (e.g., Cas12i2) that replaces amino acids in the reference Cas12i nuclease that interact with PAM is a positively charged amino acid (such as R, H or K).
  • a reference Cas12i nuclease e.g., Cas12i2
  • the engineered Cas12i nuclease comprises one, two, three, four, five, six, seven, eight or more substitutions of said amino acid residues.
  • the amino acid that interacts with PAM is an amino acid that is within 9 angstroms of the three-dimensional structure of PAM, for example: an amino acid that is within 9 angstroms of the three-dimensional structure of PAM, and within 9 angstroms of PAM.
  • Amino acids within 8 angstroms in the three-dimensional structure amino acids within 7 angstroms in the three-dimensional structure of PAM, within 6 angstroms of the three-dimensional structure of PAM, and within 5 angstroms of the three-dimensional structure of PAM
  • Amino acids within 4 angstroms in the three-dimensional structure of PAM within 3 angstroms of the three-dimensional structure of PAM, within 2 angstroms of the three-dimensional structure of PAM, or closer amino acids .
  • the distance between PAM and amino acid in spatial structure is defined by the distance between atoms in the 3D structure (PDB file) of the analyzed Cas protein-RNA-DNA three-dimensional complex, and the mutual distance between atoms can be identified by PDB file
  • the software displays In some embodiments, the spatial structural distance between the PAM and the amino acid is defined by the minimum distance between the amino acid residue and the atoms comprised by the nucleotide.
  • Programs that can be used to measure the distance between PAM and amino acids in the spatial structure, or PDB file recognition software are widely known in the art, including but not limited to PyMOL, ChimeraX, Swiss-pdbviewer, etc.
  • the one or more mutations that replace the PAM-interacting amino acid in the reference Cas12i nuclease with a positively charged amino acid are mutations at one or more of the following positions: 176, 178 , 226, 227, 229, 237, 238, 264, 447 and 563. In some embodiments, the one or more mutations that replace the PAM-interacting amino acid in the reference Cas12i nuclease with a positively charged amino acid are mutations at one or more of the following amino acids: E176, E178, Y226 , A227, N229, E237, K238, K264, T447 and E563.
  • the one or more mutations that replace the PAM-interacting amino acid in the reference Cas12i nuclease with a positively charged amino acid are mutations at one or more of the following amino acids: E176, K238, T447 and E563.
  • the mutation that replaces the amino acid that interacts with PAM in the reference Cas12i nuclease with a positively charged amino acid is located at amino acid residue 563, such as E563.
  • the amino acid position is as defined in the corresponding amino acid position of the wild-type Cas12i nuclease shown in SEQ ID NO: 1.
  • the engineered Cas12i nuclease comprises a mutation of one or more of the following amino acids: E176R, E178R, Y226R, A227R, N229R, E237R, K238R, K264R, T447R and E563R, the amino acid positions are as follows Defined by the corresponding amino acid positions of the wild-type Cas12i nuclease shown in SEQ ID NO: 1.
  • E176 is; in the cited amino acid sequence (such as relative to SEQ ID NO: 1), No. 176 amino acid E (glutamic acid); here, the common amino acid and its three
  • the letters and one-letter abbreviations are listed as follows: Alanine Ala A; Arginine Arg R; Aspartate Asp D; Cysteine Cys C; Glutamine Gln Q; Glutamate Glu E; Amino acid His H; Isoleucine Ile I; Glycine Gly G; Asparagine Asn N; Leucine Leu L; Lysine Lys K; Methionine Met M; Phenylalanine Phe F; Proline Acid Pro P; Serine Ser S; Threonine Thr T; Tryptophan Trp W; Tyrosine Tyr Y; Valine Val V.
  • the mutation of replacing the amino acid interacting with PAM in the reference Cas12i nuclease with a positively charged amino acid is to replace the corresponding amino acid residue in the reference Cas12i nuclease with R, H or K , such as R or K. In some embodiments, the mutation of replacing the amino acid interacting with PAM in the reference Cas12i nuclease with a positively charged amino acid is to replace the corresponding amino acid residue in the reference Cas12i nuclease with R.
  • the engineered Cas12i nuclease comprises one or more amino acid mutations at the following sites: 176R, 238R, 447R, and 563R, wherein the amino acid position is the wild type shown in SEQ ID NO: 1
  • the corresponding amino acid positions of the Cas12i nuclease are defined.
  • the engineered Cas12i nuclease comprises one or more of the following mutations based on the reference Cas12i nuclease: E176, K238, T447 and E563; wherein the amino acid position numbering is as shown in SEQ ID NO: 1 defined by the corresponding amino acid positions.
  • the engineered Cas12i nuclease comprises one or more of the following mutations based on the reference Cas12i nuclease: E176R, K238R, T447R and E563R; wherein the amino acid position numbering is as shown in SEQ ID NO: 1 defined by the corresponding amino acid positions.
  • the engineered Cas12i nuclease comprises an E563R mutation; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO:1.
  • the above-mentioned engineered Cas12i nuclease (replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positively charged amino acids) have at least about 85% (e.g., any at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99%) sequence identity engineered Cas12i nuclease.
  • the engineered Cas12i nuclease comprises any mutation or combination of mutations at the following amino acid residue positions: (i) 176, 238, 264, 447, 563, 176+238, 176+447, 176 +563, 238+447, 238+563, 447+563, 176+238+447, 176+238+563, 176+447+563, 238+447+563, 176+238+447+563; among them, amino acid Position numbers are as defined for the corresponding amino acid positions shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease comprises any one of the following mutations/mutation combinations: E176R, K238R, E264R, T447R, E563R, E176R+K238R, E176R+T447R, E176R+E563R, K238R+T447R, K238R+E563R, T447R+E563R, E176R+K238R+T447R, E176R+K238R+E563R, E176R+T447R+E563R, K238R+T447R+E563R, and E176R+K238R+T447R+E563R; wherein, the amino acid position NO ID number is as follows : Defined by the corresponding amino acid position shown in 1.
  • the engineered Cas12i nuclease comprises any one of the following mutations/mutation combinations: E563R, E176R+T447R, E176R+E563R, K238R+E563R, E176R+K238R+T447R, E176R+K238R+E563R, E176R+T447R+E563R, and E176R+K238R+T447R+E563R; wherein, the amino acid position numbers are as defined in the corresponding amino acid positions shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease comprises a combination of E176R+K238R+T447R+E563R mutations; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO:1.
  • an engineered Cas12i nuclease (replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with a positively charged amino acid) with at least about 85 % (eg, any at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence Consensus engineered Cas12i nuclease.
  • the engineered Cas12i nuclease comprises one or more mutations based on a reference Cas12i nuclease (e.g., Cas12i2), wherein the mutation is one of the reference Cas12i nucleases involved in opening the DNA double strand. Or multiple amino acids are replaced by amino acids with aromatic rings (such as F, Y or W). In some embodiments, the engineered Cas12i nuclease comprises one, two, three, four, five, six, or more substitutions of said amino acid residues.
  • a reference Cas12i nuclease e.g., Cas12i2
  • the engineered Cas12i nuclease comprises one, two, three, four, five, six, or more substitutions of said amino acid residues.
  • the one or more amino acids involved in opening the DNA double strand are the amino acids that interact with the last base pair at the 3' end of the PAM relative to the target strand.
  • the PAM sequence recognized by Cas12i2 is 5'-NTTN-3' base pair, wherein the base pair formed by the N base at the 3' end of the PAM sequence and the target strand is the "relative to the PAM" described in the text The last base pair at the 3' end of the target strand," followed by the sequence of the targeting site.
  • the one or more amino acids involved in opening the DNA double strand are located at the following positions: 163 and/or 164; wherein the amino acid position numbers are as defined in the corresponding amino acid positions shown in SEQ ID NO:1.
  • the one or more amino acids involved in opening the DNA double strand are one or more of the following amino acids: Q163, N164; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO: 1 .
  • the amino acid involved in opening the double-strand of DNA is N164; wherein the numbering of the amino acid position is as defined in the corresponding amino acid position shown in SEQ ID NO:1.
  • the amino acid involved in opening the double strand of DNA is replaced by F, Y or W. In some embodiments, the amino acid involved in opening the double strand of DNA is replaced by F. In some embodiments, the amino acid involved in opening the double strand of DNA is replaced by Y.
  • the engineered Cas12i nuclease comprises a mutation of any one or more of the following amino acid residues: 163F, 163Y, 163W, 164W, 164F or 164Y; wherein the amino acid position numbering is as shown in SEQ ID NO: 1 defined by the corresponding amino acid positions indicated.
  • the engineered Cas12i nuclease comprises a mutation of any one or more of the following amino acid residues: 163F, 163Y, 163W, 164F or 164Y; wherein the amino acid position numbering is as shown in SEQ ID NO: 1 The corresponding amino acid positions are defined.
  • the engineered Cas12i nuclease comprises any of the following mutations: Q163 and/or N164; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO:1. In some embodiments, the engineered Cas12i nuclease comprises any one of the following mutations: Q163F, Q163Y, Q163W, N164W, N164F or N164Y; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO: 1 .
  • the engineered Cas12i nuclease comprises any one of the following mutations: Q163F, Q163Y, Q163W, N164F or N164Y; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease comprises N164Y or N164F mutation; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease comprises an N164Y mutation; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO:1.
  • the above-mentioned engineered Cas12i nuclease (one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease are replaced with an aromatic ring amino acids) have at least about 85% (e.g., any at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity engineered Cas12i nuclease.
  • the Cas12i nuclease of described engineering comprises one or more mutations based on a reference Cas12i nuclease (eg, Cas12i2), which is located in the RuvC domain in the reference Cas12i enzyme and combined with a single-stranded Amino acids that interact with the DNA substrate are replaced with positively charged amino acids (such as R, H, or K).
  • the engineered Cas12i enzyme comprises one, two, three, four, five, six or more substitutions of said amino acid residues.
  • the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are amino acids within 9 Angstroms of the distance from the single-stranded DNA substrate on the three-dimensional structure, for example, it can be: on the three-dimensional structure Amino acids within 8 angstroms of the single-stranded DNA substrate, within 7 angstroms of the three-dimensional structure of the single-stranded DNA substrate, within 6 angstroms of the three-dimensional structure of the single-stranded DNA substrate, Amino acids within 5 angstroms from the single-stranded DNA substrate on the three-dimensional structure, amino acids within 4 angstroms from the single-stranded DNA substrate on the three-dimensional structure, and 3 angstroms away from the single-stranded DNA substrate on the three-dimensional structure Amino acids within 2 Angstroms, or amino acids closer to the single-stranded DNA substrate in the three-dimensional structure.
  • the RuvC domain is the enzymatic domain responsible for cutting single-stranded DNA or double-stranded DNA in the Cas12i protein.
  • the RuvC domain of Cas12i is divided into three parts: RuvC-1, RuvC-2 and RuvC-3. These three parts are adjacent in the three-dimensional structure and together form a catalytic pocket with enzymatic cleavage activity.
  • the three-dimensional crystal structure of Cas12i2, its domain composition, and the description of its interaction with DNA substrates are described in Huang X. et al., Nature Communications, 11, Article number: 5241 (2020).
  • the three-dimensional crystal structure of Cas12i1, its domain composition, and the description of its interaction with DNA substrates are described in Zhang H.
  • the three-dimensional structural model of the interaction between the reference Cas12i and the substrate can be obtained through homology structure comparison and homology modeling through the known three-dimensional crystal structure of Cas12i.
  • a modeling approach is described in Example 3 to obtain amino acids in Cas12i2 that are located in the RuvC domain and within 9 Angstroms of the single-stranded DNA substrate.
  • the distance between the amino acids in the RuvC domain and the single-stranded DNA substrate in the spatial structure can be determined by the atoms in the 3D structure (PDB file) of the analyzed Cas protein-RNA-DNA three-dimensional complex Defined by the distance, the mutual distance between atoms can be displayed by the PDB file recognition software.
  • the spatial structural distance of amino acids in the RuvC domain from the single-stranded DNA substrate is defined by the minimum distance between amino acid residues and atoms comprised by nucleotides.
  • PDB file recognition software Programs that can be used to measure the spatial structure distance between the amino acids in the RuvC domain and the single-stranded DNA substrate, or PDB file recognition software are widely known in the art, including but not limited to PyMOL, ChimeraX, Swiss-pdbviewer, etc.
  • the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are one or more amino acids at the following positions: 323, 327, 355, 359, 360, 361, 362, 388, 390, 391, 392, 393, 414, 417, 418, 421, 424, 425, 650, 652, 653, 696, 705, 708, 709, 751, 752, 755, 840, 848, 851, 856, 885, 897, 925, 926, 928, 929, 932, and 1022.
  • the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are one or more of the following amino acids: E323, L327, V355, G359, G360, K361, D362, L388, N390, N391, F392, K393, Q414, L417, L418, K421, Q424, Q425, S650, E652, G653, I696, K705, K708, E709, L751, S752, E755, N840, N848, S851, A856, Q885, M897, N925, I926, T928, G929, Y932, A1022.
  • the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are one or more of the following amino acids: E323, D362, L388, N391, L417, Q424, Q425, N925, I926, and G929.
  • the amino acid position is as defined in the corresponding amino acid position of the wild-type Cas12i nuclease shown in SEQ ID NO: 1.
  • the engineered Cas12i nuclease comprises replacing one or more amino acids in the reference Cas12i nuclease that are located in the RuvC domain and interact with single-stranded DNA substrates with R, H or K (e.g. R or K) mutations.
  • the engineered Cas12i nuclease comprises a mutation in which one or more amino acids located in the RuvC domain and interacting with a single-stranded DNA substrate in the reference Cas12i nuclease are replaced with R.
  • the engineered Cas12i nuclease comprises one or more of the following amino acid mutations or mutation combinations: N390R, N391R, F392R, L751R, E755R, N840R, N848R, S851R, A856R, Q885R, M897R, I926R ⁇ G929R ⁇ Y932R ⁇ E323R ⁇ L327R ⁇ V355R ⁇ G359R ⁇ G360R ⁇ K361R ⁇ D362R ⁇ Q414R ⁇ K421R ⁇ Q425R ⁇ S650R ⁇ E652R ⁇ K705R ⁇ K708R ⁇ E709R ⁇ S752R ⁇ N925R ⁇ T928R ⁇ E323R+D362R ⁇ E323R+Q425R ⁇ E323R +I926R ⁇ Q425R+I926R ⁇ E323R+D362R+Q425R ⁇ E323R+I926R ⁇ Q425R+
  • the engineered Cas12i nuclease comprises a mutation or a combination of mutations at any one of the following amino acid residue positions: E323, D362, Q425, N925, I926 and G929; wherein, the amino acid position numbering is as SEQ ID NO: Defined by the corresponding amino acid positions shown in 1.
  • the engineered Cas12i nuclease comprises a mutation or combination of mutations at any one of the following amino acid residue positions: E323, D362, Q425, N925, I926, E323+D362, E323+Q425, E323+I926, D362+Q425, D362+N925, D362+I926, Q425+I926, N925+I926, E323+D362+Q425, E323+D362+I926, E323+Q425+I926, D362+N925+I926, D362+Q425+I926, E323+D362+Q425+I926; wherein, the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO:1.
  • the mutation is a mutation that replaces the amino acid residue at the position with R, H, or K (such as R).
  • the engineered Cas12i nuclease comprises any one or amino acid combination of the following: 323R, 362R, 425R, 925R, 926R, 323R+362R, 323R+425R, 323R+926R, 362R+425R, 362R +926R, 425R+926R, 925R+926R, 323R+362R+425R, 323R+362R+926R, 323R+425R+926R, 362R+925R+926R, 323R+362R+425R+926R, and 362R+425R+926R; Wherein, the amino acid position number is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease comprises any one of the following mutations or mutation combinations: E323R, D362R, Q424R, Q425R, N925R, I926R, and G929R; wherein, the amino acid position numbering is as shown in SEQ ID NO: 1 defined by the corresponding amino acid positions indicated.
  • the engineered Cas12i nuclease comprises any one of the following mutations or combinations of mutations: E323R, D362R, Q425R, N925R, I926R, E323R+D362R, E323R+Q425R, E323R+I926R, D362R+Q425R, Q425R +I926R, D362R+I926R, N925R+I926R, E323R+D362R+Q425R, E323R+D362R+I926R, E323R+Q425R+I926R, D362R+N925R+I926R, D362R+Q425R+I926R, and E9323R+
  • the amino acid position number is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease comprises an I926R mutation; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO:1. In some embodiments, the engineered Cas12i nuclease comprises an E323R+D362R mutation; wherein, the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO:1. In some embodiments, in order to achieve the purpose of improving gene editing efficiency, one or more combinations with the above-mentioned engineered Cas12i nuclease (the reference Cas12i nuclease is located in the RuvC domain and interacts with the single-stranded DNA substrate) can also be used.
  • replacement of amino acids with positively charged amino acids has at least about 85% (e.g., any at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%) sequence identity engineered Cas12i nuclease.
  • the engineered Cas12i nuclease comprises one or more mutations based on a reference Cas12i nuclease (e.g., Cas12i2) that interacts with the DNA-RNA duplex in the reference Cas12i nuclease
  • a reference Cas12i nuclease e.g., Cas12i2
  • One or more amino acids that act are replaced with positively charged amino acids (such as R, H, or K).
  • the engineered Cas12i enzyme comprises one, two, three, four, five, six, or more substitutions of said amino acid residues.
  • the one or more amino acids interacting with the DNA-RNA double helix are amino acids within 9 Angstroms of the distance from the DNA-RNA double helix on the three-dimensional structure, for example, it can be: on the three-dimensional structure and the DNA-RNA double helix Amino acids whose helical distance is within 8 angstroms, amino acids whose three-dimensional structure is within 7 angstroms of DNA-RNA double helix, amino acids whose three-dimensional structure is within 6 angstroms of DNA-RNA double helix, and three-dimensional structure Amino acids within 5 angstroms of DNA-RNA double helix distance, within 4 angstroms of DNA-RNA double helix distance in three-dimensional structure, within 3 angstroms of DNA-RNA double helix distance in three-dimensional structure, in Amino acids that are within 2 Angstroms of the distance from the DNA-RNA double helix in the three-dimensional structure, or amino acids that are closer.
  • Cas nucleases work as follows: Cas forms a complex with a guide RNA (such as crRNA), where the crRNA and the target DNA pair to form a DNA-RNA double helix, and interact with the Cas nuclease to open the double-stranded target. To the DNA, and form R-loop, so that the Cas enzyme cutting active site completes the cutting of dsDNA.
  • a guide RNA such as crRNA
  • the crRNA and the target DNA pair to form a DNA-RNA double helix
  • the Cas enzyme cutting active site completes the cutting of dsDNA.
  • the three-dimensional crystal structure of Cas12i2, its domain composition, and the description of its interaction with the DNA-RNA double helix are described in Huang X. et al., Nature Communications, 11, Article number: 5241 (2020).
  • the distance between the DNA-RNA double helix and the Cas amino acid in the spatial structure can be determined by the amino acid residues and nucleotides in the 3D structure (PDB file) of the analyzed Cas protein-RNA-DNA three-dimensional complex The minimum distances between included atoms are defined, and the mutual distances between atoms can be displayed by PDB file recognition software.
  • the distance between the Cas amino acid and the DNA-RNA double helix in the spatial structure is defined by the distance between the hypothetical positions of the atoms.
  • the one or more amino acids that interact with the DNA-RNA duplex are one or more amino acids at the following positions: 116, 117, 156, 159, 160, 161, 247, 293, 294 ,297,301,305,306,308,312,313,316,319,320,343,348,349,427,433,438,441,442,679,683,691,782,783,797,800 , 852, 853, 855, 861, 865, 957, 958.
  • the one or more amino acids that interact with the DNA-RNA duplex are one or more of the following amino acids: G116, E117, A156, T159, E160, S161, E247, G293, E294, N297 , T301, I305, K306, T308, N312, F313, Q316, E319, Q320, E343, E348, E349, D427, K433, V438, N441, Q442, N679, E683, E691, D782, E783, E797, E800, M852 , D853, L855, N861, Q865, S957, D958.
  • the one or more amino acids that interact with the DNA-RNA duplex are one or more of the following: G116, E117, T159, S161, E319, E343, or D958.
  • the amino acid that interacts with the DNA-RNA duplex is D958.
  • the amino acid position is as defined in the corresponding amino acid position of the wild-type Cas12i nuclease shown in SEQ ID NO: 1.
  • the engineered Cas12i nuclease comprises one or more amino acids that interact with the DNA-RNA duplex in the reference Cas12i nuclease are replaced by R, H or K (such as R or K). mutation. In some embodiments, the engineered Cas12i nuclease comprises a mutation that replaces one or more amino acids involved in the DNA-RNA double helix interaction in the reference Cas12i nuclease with R.
  • the engineered Cas12i nuclease comprises one or more of the following amino acid mutations: G116R, E117R, A156R, T159R, S161R, T301R, I305R, K306R, T308R, N312R, F313R, D427R, K433R, V438R ⁇ N441R ⁇ Q442R ⁇ M852R ⁇ L855R ⁇ N861R ⁇ Q865R ⁇ E160R ⁇ Q316R ⁇ E319R ⁇ Q320R ⁇ E247R ⁇ E343R ⁇ E348R ⁇ E349R ⁇ N679R ⁇ E683R ⁇ E691R ⁇ D782R ⁇ E783R ⁇ E797R ⁇ E800R ⁇ D853R ⁇ S957R ⁇ D958R ⁇ G293R, E294R, N297R; Described amino acid position is defined as the corresponding amino acid position of the wild-type Cas12i nuclease shown in
  • the engineered Cas12i nuclease comprises a mutation or a combination of mutations at any one of the following amino acid residue positions: G116, E117, T159, S161, E319, E343, or D958; wherein, the amino acid position numbering is as in SEQ Defined by the corresponding amino acid position shown in ID NO:1.
  • the mutation is a mutation that replaces the amino acid residue at the position with R, H or K (eg, R).
  • the engineered Cas12i nuclease comprises any one of the following site amino acids or amino acid mutation combinations: 116R, 117R, 159R, 161R, 319R, 343R, or 958R; wherein, the amino acid position numbering is as SEQ ID NO : Defined by the corresponding amino acid position shown in 1.
  • the engineered Cas12i nuclease comprises any one of the following mutations or mutation combinations: G116R, E117R, T159R, S161R, E319R, E343R, or D958R; wherein, the amino acid position numbering is as shown in SEQ ID NO: 1 defined by the corresponding amino acid positions indicated.
  • the engineered Cas12i nuclease comprises a D958R mutation; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO:1.
  • the above-mentioned engineered Cas12i nuclease has at least about 85% (such as any at least about 86%, 87%, 88%, 89%, 90%) %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity engineered Cas12i nuclease.
  • the engineered Cas12i nuclease comprises one or more mutations based on a reference Cas12i nuclease (e.g., Cas12i2) that interacts with the DNA-RNA duplex in the reference Cas12i nuclease
  • a reference Cas12i nuclease e.g., Cas12i2
  • One or more polar or positively charged amino acids are replaced with hydrophobic amino acids (such as A, V, I, L, M, F, Y, P, C or W).
  • the mutation also referred to herein as a "high specificity” or "HF” mutation
  • the off-target rate of CasXX-HF can be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% relative to CasXX or lower even more.
  • the engineered Cas12i enzyme comprises one, two, three, four, five, six or more substitutions of said amino acid residues.
  • the one or more amino acids interacting with the DNA-RNA double helix are amino acids within 9 Angstroms of the distance from the DNA-RNA double helix on the three-dimensional structure, for example, it can be: on the three-dimensional structure and the DNA-RNA double helix Amino acids whose helical distance is within 8 angstroms, amino acids whose three-dimensional structure is within 7 angstroms of DNA-RNA double helix, amino acids whose three-dimensional structure is within 6 angstroms of DNA-RNA double helix, and three-dimensional structure Amino acids within 5 angstroms of DNA-RNA double helix distance, within 4 angstroms of DNA-RNA double helix distance in three-dimensional structure, within 3 angstroms of DNA-RNA double helix distance in three-dimensional structure, in Amino acids that are within 2 Angstroms of the distance from the DNA-RNA double helix in the three-dimensional structure, or amino acids that are closer.
  • the one or more polar or positively charged amino acids that interact with the DNA-RNA duplex are one or more of the following positions: 119, 164, 297, 308, 309, 312, 346, 357, 394, 395, 402, 441, 433, 565, 715, 719, 766, 782, 807, 841, 844, 845, 848, 857, 861, and 865.
  • the one or more polar or positively charged amino acids that interact with the DNA-RNA duplex are one or more of the following: Y119, Y164, N297, T308, R309, N312, S346, H357, K394, E395, R402, N441, K433, S565, R715, R719, S766, D782, K807, N841, K844, K845, N848, R857, N861 and Q865.
  • the one or more polar or positively charged amino acids that interact with the DNA-RNA duplex are one or more of the following: R857, R861, K807, N848, R715, R719, K394, H357 and K844.
  • the one or more polar or positively charged amino acids that interact with the DNA-RNA duplex are one or more of the following: R857, R719, K394, and K844.
  • the amino acid position as described above is defined as the corresponding amino acid position shown in SEQ ID NO: 1 or 8.
  • the engineered Cas12i nuclease comprises one or more mutations: S565A, N297A, Q865A, T308A, R309A, N312A, N441A, R857A, N861A, Y119F, K433A, K807A, N841A, N848A , K845A, D782A, R715A, R719A, S766A, K394A, H357A, K844A, E395A, S346A, R402A, Y164N; wherein, the amino acid position numbers are as defined in the corresponding amino acid positions shown in SEQ ID NO: 1 or 8.
  • the Cas12i nuclease of described engineering comprises will reference Cas12i nuclease located in 357,394,715,719,807,844,848,857,861 one or more polar or positive Substitution of charged amino acids for mutations of hydrophobic amino acids.
  • the hydrophobic amino acid is selected from A, V, L, I, P and F, such as A, V, L or I.
  • the engineered Cas12i nuclease comprises one or more polarities or A mutation in which a positively charged amino acid is replaced by an A.
  • the amino acid position numbers are as defined in the corresponding amino acid positions shown in SEQ ID NO: 1 or 8.
  • the engineered Cas12i nuclease comprises a mutation of any one of the following amino acid or amino acid combinations: H357A, K394A, R715A, R719A, K807A, K844A, N848A, R857A, and R861A; wherein, the amino acid position numbering As defined by the corresponding amino acid position shown in SEQ ID NO: 1 or 8.
  • the engineered Cas12i nuclease comprises a mutation or combination of mutations at any one of the following amino acid residue positions: 857, 719, 394, or 844; wherein, the amino acid position numbering is as shown in SEQ ID NO: 1 defined by the corresponding amino acid positions.
  • the mutation is a mutation that replaces the amino acid residue at the position with a hydrophobic amino acid (eg, A).
  • the engineered Cas12i nuclease comprises a mutation at any one of the following amino acid positions or amino acid combinations: R857, R719, K394, or K844; wherein, the amino acid position numbering is as shown in SEQ ID NO: 1 or 8 defined by the corresponding amino acid positions.
  • the engineered Cas12i nuclease comprises a mutation or a combination of mutations at any one of the following amino acid residue positions: R857, R719, K394, K844, R719+K394, K394+K844, R857+K394, R719+ K844, R857+R719, R857+K844, R719+K394+K844, R857+R719+K394, R857+K394+K844, R857+R719+K844, R857+R719+K844, R857+R719+K844, R857+R719+K394+K844; wherein, the amino acid position numbering is as SEQ ID NO: defined by the corresponding amino acid position shown in 1 or 8; the mutation is a mutation in which the amino acid residue at the position is replaced by a hydrophobic amino acid (such as A).
  • the engineered Cas12i nuclease comprises a mutation or a combination of mutations at any one of the following amino acid residue positions: R857A, R719A, K394A, K844A, R719A+K394A, K394A+K844A, R857A+K394A, R719A+ K844A, R857A+R719A, R857A+K844A, R719A+K394A+K844A, R857A+R719A+K394A, R857A+K394A+K844A, R857A+R719A+K844A, R857A+R719A+K844A, R857A+R719A+K844A, R857A+R719A+K394A+K844A, where the amino acid ID number is as SE; NO: Defined by the corresponding amino acid position indicated by 1 or 8.
  • the engineered Cas12i nuclease comprises any one of the following mutations or combinations of mutations: R857A, R719A, K394A, or K844A; wherein, the amino acid position numbering is the corresponding amino acid shown in SEQ ID NO: 1 or 8 defined by the location.
  • the engineered Cas12i nuclease comprises a K844A mutation; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO: 1 or 8.
  • the engineered Cas12i nuclease comprises R719A and K844A mutations; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO: 1 or 8. In some embodiments, the engineered Cas12i nuclease comprises R857A and K844A mutations; wherein the amino acid position numbering is as defined in the corresponding amino acid position shown in SEQ ID NO: 1 or 8.
  • the above-mentioned engineered Cas12i nuclease (with reference to one or more polarity or polarities that interact with the DNA-RNA double helix in the Cas12i nuclease).
  • a positively charged amino acid is replaced by a hydrophobic amino acid) having at least about 85% (e.g., any at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%) sequence identity engineered Cas12i nuclease.
  • the mutations described herein may include one or more of: insertions, deletions, substitutions, and may be mutations of a single amino acid or multiple amino acids.
  • Any one or more mutations described in sections 1) to 5) can be combined with any one or more known mutations that increase Cas12i activity, such as target binding, double-strand cleavage activity, nickase activity and/or gene editing activity. Exemplary mutations can be found, for example, in the following documents PCT/CN2020/0134249 and CN 112195164 A, which are incorporated herein by reference in their entirety. Any one or more mutations described in sections 1) to 5) can also be combined with any one or more known mutations that reduce Cas12i activity, such as target binding, double-strand cleavage activity, nickase activity and/or gene editing activity .
  • the engineered Cas12i nuclease (such as the engineered Cas12i nuclease comprising one or more mutations in the above 1) to 5) sections) also comprises one or more flexible region mutations, the The mutation increases (such as increases by at least about any 10%, 20%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold times, 5 times, 10 times, 20 times, 50 times, 100 times or more) reference Cas12i nuclease (or the engineered Cas12i comprising any one or more mutations in (1)-(5) sections The flexibility of the flexible region in nucleases).
  • the flexible region in the reference Cas12i nuclease can be determined using any method known in the art. In some embodiments, multiple flexible regions are determined based solely on the amino acid sequence of the reference Cas12i nuclease. In some embodiments, multiple flexible regions are determined based on structural information of the reference Cas12i nuclease, including, for example, secondary structure, crystal structure, NMR structure, and the like.
  • the method for engineering transformation Cas12i nuclease flexible region described here comprises: (a) obtains the Cas12i nuclease of multiple engineering, the Cas12i nuclease of every kind of engineering comprises one or more mutations, and said mutation increases Referring to the flexibility of the flexible region in one or more flexible regions of the Cas12i nuclease; and (b) selecting one or more engineered Cas12i nucleases from the plurality of engineered Cas12i nucleases, wherein the One or more engineered Cas12i nucleases have increased activity (eg, target binding, double-strand cleavage activity, nickase activity, and/or gene editing activity) compared to the reference Cas12i nuclease.
  • activity eg, target binding, double-strand cleavage activity, nickase activity, and/or gene editing activity
  • the method further comprises determining one or more regions of flexibility in the reference Cas12i nuclease. In some embodiments, the method further comprises measuring the activity of the engineered Cas12i nuclease in a eukaryotic cell, such as a mammalian cell (eg, a human cell).
  • a mammalian cell eg, a human cell
  • the plurality of regions of flexibility are determined using a program selected from the group consisting of: PredyFlexy, FoldUnfold, PROFbval, Flexserv, FlexPred, DynaMine, and Disomine.
  • the one or more flexible regions are located at random coils.
  • the one or more flexible regions are between the reference Cas12i nuclease (or the engineered Cas12i nuclease comprising any one or more mutations in sections (1)-(5)) and In the domain of DNA and/or RNA interaction.
  • the flexible region is at least about 5 (eg, 5) amino acids in length.
  • the one or more mutations comprise insertion of one or more (eg, 2) glycine (G) residues in the flexible region.
  • the one or more G residues are inserted at the N-terminus of the flexible amino acid residues in the flexible region, wherein the flexible amino acid residues are selected from the group consisting of G, serine (S), asparagine Amide (N), aspartic acid (D), histidine (H), methionine (M), threonine (T), glutamic acid (E), glutamine (Q), lysine ( K), arginine (R), alanine (A) and proline (P).
  • the flexible amino acid residues are selected according to the following priority: G>S>N>D>H>M>T>E>Q>K>R>A>P.
  • the one or more mutations comprise replacing one or more non-G residues with one or more G residues.
  • the one or more mutations comprise replacing a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of leucine (L), isoleucine Acid (I), Valine (V), Cysteine (C), Tyrosine (Y), Phenylalanine (F) and Tryptophan (W).
  • L leucine
  • I isoleucine Acid
  • V Valine
  • C Cysteine
  • Y Tyrosine
  • F Phenylalanine
  • W Tryptophan
  • the activity is a site-specific nuclease activity.
  • the activity is gene editing activity in a eukaryotic cell (eg, a human cell).
  • the gene editing efficiency is measured using a T7 endonuclease 1 (T7E1) assay, sequencing of target DNA, assayed by resolution tracking indels (TIDE), or by amplicon analysis Indel detection (IDAA) assays were performed.
  • T7E1 T7 endonuclease 1
  • TIDE resolution tracking indels
  • IDAA amplicon analysis Indel detection
  • the engineered Cas12i nuclease (for example, the engineered Cas12i nuclease comprising any one or more mutations in sections (1)-(5)) comprises one or more flexible region mutations
  • the flexible region mutation increases the flexibility of the flexible region in the reference Cas12i nuclease (such as Cas12i2 nuclease, or the engineered Cas12i nuclease comprising any one or more mutations in (1)-(5) sections)
  • the flexible region is selected from the group of regions corresponding to amino acid residues 228-232, amino acid residues 439-443, amino acid residues 478-482, amino acid residues 500-504, amino acid residues 775-779 and Amino acid residues 925-929, wherein the numbering of the amino acid residues is as defined in the corresponding amino acid positions shown in SEQ ID NO:1.
  • the flexible region is selected from amino acid residues 439-443 or amino acid residues 925-929, wherein the numbering of the amino acid residues is as defined in the corresponding amino acid positions shown in SEQ ID NO:1.
  • the reference Cas12i enzyme is Cas12i2 (SEQ ID NO: 1).
  • the one or more flexible region mutations comprise insertion of one or more (eg, 2) G residues in the flexible region.
  • the one or more G residues are inserted at the N-terminus of flexible amino acid residues in the flexible region, wherein the flexible amino acid residues are selected from the group consisting of G, S, N, D, H, M, T, E, Q, K, R, A, and P.
  • the flexible amino acid residues are selected according to the following priority: G>S>N>D>H>M>T>E>Q>K>R>A>P.
  • the one or more flexible region mutations comprise replacing a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: A, V, I, L, M, F, Y, P, C or W; preferably, selected from: L, I, V, C, Y, F and W.
  • the flexible region mutation is at 439 and/or 926. In some embodiments, they are one or more of the following amino acids: L439, I926. Wherein, the amino acid position is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease (for example, the engineered Cas12i nuclease comprising any one or more mutations in sections (1)-(5)) comprises 926G and/or 439(L +G) mutation.
  • the engineered Cas12i nuclease comprises one or more of the following flexibility region mutations: I926G, L439(L+G), and L439(L+GG).
  • the engineered Cas12i nuclease comprises an I926G mutation.
  • the engineered Cas12i nuclease comprises a L439(L+G) mutation.
  • the engineered Cas12i nuclease comprises a L439(L+GG) mutation.
  • the amino acid residue numbering is based on SEQ ID NO:1.
  • L439(L+G) is that in the cited amino acid sequence (such as SEQ ID NO: 1), a glycine (G) is inserted after the 439th amino acid, the original 439th
  • the L sequence of bits is unchanged; in the context of this application and in the drawings, it is also sometimes indicated as 439G.
  • L439 (L+GG) is that in the cited amino acid sequence, two glycines (GG) are inserted after the 439th amino acid, and the original 439th L sequence remains unchanged; in the context of this application And in the drawings, it is sometimes expressed as 439GG.
  • the engineered Cas12i nuclease comprises any one of the following amino acid residue positions Mutation or combination of mutations: 926, 439, 925+926, 362+925+926, 439+926, 323+362+926 (such as I926, L439, N925+I926, D362+N925+I926, L439+I926, E323 +D362+I926); Wherein, the amino acid position numbering is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • the mutation at amino acid position 323, 362, 925 or 926 is a mutation that replaces the amino acid residue at the position with R, H or K (such as R); wherein, the amino acid position numbering is as in SEQ ID NO:1 is defined by the corresponding amino acid position.
  • the mutation at amino acid position 439 or 926 is a mutation that replaces the amino acid residue at the position with G or inserts G or GG after the amino acid residue; wherein, the amino acid position numbering is as in SEQ ID NO:1 is defined by the corresponding amino acid position.
  • the engineered Cas12i nuclease (for example, the engineered Cas12i nuclease comprising any one or more mutations in sections (1)-(5)) comprises any of the following amino acid residues or Amino acid residue combination mutation: 926G, 439(L+GG), 925R+926G, 362R+925R+926G, 439(L+GG)+926R, or 323R+362R+926G; wherein, the amino acid position number is as in SEQ ID NO:1 is defined by the corresponding amino acid position.
  • the engineered Cas12i nuclease comprises any one of the following mutations or combination of mutations: I926G, L439(L+GG), L439(L+GG)+I926R, N925R+I926G, D362R+N925R+I926G , E323R+D362R+I926G; wherein the amino acid position numbers are as defined in the corresponding amino acid positions shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease (for example, the engineered Cas12i nuclease comprising any one or more mutations in (1)-(5) sections) Cas12i nuclease, and/or the engineered Cas12i nuclease comprising above-mentioned flexible region mutation) has at least about 85% (such as any at least about 86%, 87%, 88%, 89%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity engineered Cas12i nuclease.
  • the engineered Cas12i nuclease has at least about 85% (such as any at least about 86%, 87%, 88%, 89%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity engineered Cas12i nuclease.
  • the engineered Cas12i nuclease comprises a mutation or combination of mutations at any one of the following amino acid residue positions: 164, 176, 238, 323, 357, 362, 394, 439, 447, 563, 715 ,719,807,844,848,857,861,925,926,958,176+238+447+563,323+362,176+238+447+563+164,176+238+447+563+926 , 176+238+447+563+323+362, 164+926, 164+323+362, 176+238+447+563+164+926, 176+238+447+563+164+323+362, 176 +238+447+563+164+926+323+362, 176+238+447+563+164+323+362, 176+238+447+563+164+323+362, 176
  • the mutation at amino acid position 176, 238, 323, 362, 447, 563, 926, or 958 is a mutation that replaces the amino acid residue at the position with R, H or K (such as R) .
  • the mutation at amino acid position 164 is a mutation that replaces the amino acid residue at that position with Y or F (eg, Y).
  • the mutation at amino acid position 439 or 926 is a mutation that replaces the amino acid residue at the position with G or inserts G or GG after the amino acid residue.
  • the engineered Cas12i nuclease comprises a mutation of any one of the following amino acid residues or combinations of amino acid residues: N164, E176; K238; E323, D362, T447; E563, I926, I926, D958, L439 , R857, N861, K807, N848, R715, R719, K394, H357, K844, E176+K238+T447+E563, E323+D362, E176+K238+T447+E563+N164, E176+K238+T447+E563+I926 , E176+K238+T447+E563+E323+D362, N164+I926, N164+E323+D362, E176+K238+T447+E563+N164+I926, E176+K238+T447+E563+N164+I926, E176+K238+T447+E56
  • amino acid position number is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease comprises any one of the following mutations or combination of mutations:
  • amino acid position number is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease comprises the following combination of mutations:
  • E176R+K238R+T447R+E563R+N164Y+E323R+D362R wherein the amino acid position number is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • the mutant is named as CasXX hereinafter, and its sequence number is SEQ ID NO: 8.
  • the engineered Cas12i nuclease comprises any one of the following mutation combinations:
  • the engineered Cas12i nuclease comprises the following combination of mutations:
  • the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, and D362R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, and I926R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, E323R, and D362R mutations.
  • the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, and I926G mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G, and L439 (L+GG) mutations.
  • the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G, and L439 (L+G) mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, and D958R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, I926R, and D958R mutations.
  • the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, and D958R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, I926R, E323R, D362R, and D958R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G, and D958R mutations.
  • the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G, L439 (L+GG), and D958R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, and K844A mutations.
  • the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, R719A, and K844A mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, R857A, and K844A mutations.
  • the amino acid position numbers are as defined in the corresponding amino acid positions shown in SEQ ID NO:1.
  • Cas12i nuclease with the above-mentioned engineered Cas12i nuclease has at least about 80% (such as any at least about 81%, 82%, 83%, 84%, 85%) %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity engineering Cas12i nuclease.
  • an engineered Cas12i nuclease which comprises any amino acid sequence shown in SEQ ID NOs: 2 ⁇ 24, or has any amino acid sequence shown in SEQ ID NOs: 2 ⁇ 24
  • the amino acid sequence has at least about 80% (e.g., any at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% %, 94%, 95%, 96%, 97%, 98%, 99%) amino acid sequence identity.
  • the reference Cas12i nuclease is Cas12i1, Cas12i2, or an ortholog thereof. In some embodiments, the reference Cas12i nuclease is native Cas12i1, or a variant thereof (eg, a naturally occurring variant). In some embodiments, the reference Cas12i nuclease is native Cas12i2 (as shown in SEQ ID NO: 1), or a variant thereof (such as a naturally occurring variant). In some embodiments, the reference Cas12i nuclease is an engineered Cas12i nuclease (the engineered Cas12i comprising any one or more mutations in sections (1)-(5) as described in the present invention Nuclease). In some embodiments, the reference Cas12i nuclease is CasXX (SEQ ID NO: 8).
  • Type V-I CRISPR-Cas12i has been identified as an RNA-guided DNA endonuclease system. Unlike CRISPR-Cas systems such as Cas12b or Cas9, the Cas12i-based CRISPR system does not require a tracrRNA sequence.
  • the RNA guide sequence comprises crRNA.
  • the crRNAs described herein include direct repeat sequences and spacer sequences.
  • the crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or a spacer sequence.
  • the crRNA includes a direct repeat sequence, a spacer sequence, and a direct repeat sequence (DR-spacer-DR), which is typical of precursor crRNA (pre-crRNA) configurations in other CRISPR systems .
  • the crRNA includes truncated direct repeat and spacer sequences, which are typical features of processed or mature crRNA.
  • the CRISPR-Cas12i effector protein forms a complex with an RNA guide sequence, and the spacer sequence directs the complex to sequence-specific binding to a target nucleic acid that is complementary to the spacer sequence (eg at least 70% complementary).
  • the engineered Cas12i of the present application is an endonuclease that binds to a specific site of a target sequence and cuts under the guidance of a guide RNA, and has DNA and RNA endonuclease activity.
  • the Cas12i is capable of autonomous crRNA biogenesis by processing a precursor crRNA array. Autonomous pre-crRNA processing facilitates Cas12i delivery, enabling double-nicking applications, as two separate genomic loci can be targeted by a single crRNA transcript.
  • the Cas12i protein then processes the CRISPR array into two homologous crRNAs, forming a paired nicking complex.
  • the guide RNA comprises a precursor crRNA expressed from a CRISPR array consisting of target sequences interleaved with unprocessed DR sequences, repeated by intrinsic precursor crRNA processing of the effector protein to enable simultaneous targeting of one, two or more sites.
  • Cas12i nucleases from various organisms can be used as the reference Cas12i nucleases to provide the engineered Cas12i nucleases and effector proteins of the present application.
  • Exemplary Cas12i nucleases have been described, eg, in WO2019/201331A1 and US2020/0063126A1, which are hereby incorporated by reference in their entirety.
  • the reference Cas12i nuclease has enzymatic activity.
  • the reference Cas12i is a nuclease, ie, cleaves both strands of a target duplex nucleic acid (eg, duplex DNA).
  • the reference Cas12i is a nickase, ie, cuts a single strand of a target duplex nucleic acid (eg, duplex DNA).
  • the reference Cas12i nuclease is enzymatically inactive.
  • the reference Cas12i nuclease is Cas12i1, Cas12i2, or Cas12i-Phi.
  • the reference Cas12i nuclease contains the sequence of SEQ ID NO: 1.
  • the engineered Cas12i nuclease is based on a functional variant (or functional derivative) of a naturally occurring Cas12i nuclease.
  • a functional variant or functional derivative
  • a functional variant differs in amino acid sequence by at least one amino acid residue (eg, with a deletion, insertion, substitution and/or fusion).
  • the functional variant has one or more mutations, such as amino acid substitutions, insertions and deletions.
  • the functional variant may comprise 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10 or more amino acid substitutions.
  • the one or more substitutions are conservative substitutions.
  • the functional variant has all domains of a naturally occurring Cas12i nuclease.
  • the functional variant does not have one or more domains of a naturally occurring Cas12i nuclease.
  • the functional variant has all domains of the engineered Cas12i nuclease.
  • the functional variant does not have one or more domains of the engineered Cas12i nuclease.
  • the biological activity of the functional variant of the Cas12i nuclease changes due to a change in its amino acid, for example, from a native nuclease to an enzyme-inactive mutant.
  • the Cas12i variant may comprise Domain, identity percentage, etc.) Cas12i protein sequence.
  • the functional variant of the engineered Cas12i nuclease has enzymatic activity (such as DNA double-strand or single-strand cleavage activity), or at least has a reference Cas12i nuclease (or its parent engineered Cas12i nucleic acid Enzyme) Enzyme activity of about 60%, such as at least about any of 65%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the enzymatic activity of the functional variant of the engineered Cas12i nuclease is at least 1.1 times (such as at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5-fold, 10-fold or more) of the enzyme activity.
  • the functional variant of the engineered Cas12i nuclease has a different catalytic activity than its engineered non-functional variant mutant form of the Cas12i nuclease.
  • the functional variant mutations eg, amino acid substitutions, insertions and/or deletions
  • the functional variant of the engineered Cas12i nuclease comprises mutations in one or more catalytic domains.
  • a Cas12i nuclease that cleaves one strand of a double-stranded target nucleic acid without cutting the other is referred to herein as a "nickase” (eg, "Cas12i nickase”).
  • a Cas12i nuclease having substantially no nuclease activity is referred to as an inactivated Cas12i protein ("dCas12i") (to which a heterologous polypeptide (fusion partner) fused thereto may provide nuclease activity, see “Engineered Cas12i” below for details. Cas12i Effector Proteins” section).
  • the DNA cleavage activity of the functional variant mutant enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or more relative to the mutant form of its non-functional variant
  • the functional variant of the Cas12i nuclease is considered to substantially lack all DNA cleavage activity.
  • the mutation of one or more amino acid residues in the Cas12i nuclease active site can lead to Cas12i (dCas12i) that reduces or loses enzymatic activity, also referred to in the present invention as " Cas12i of nuclease activity loss” or " enzymatic inactivation mutation body”.
  • the engineered Cas12i nucleases provided herein can be modified to have reduced or absent nuclease activity, e.g., Cas12i nuclease activity deficient with wild-type Cas12i nuclease (or its parent engineered Cas12i Nuclease activity (such as DNA double-strand or single-strand cleavage activity) is reduced by at least about 50% (such as a reduction of at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%) , 98%, 99%, or any of 100%).
  • Cas12i nuclease activity deficient with wild-type Cas12i nuclease or its parent engineered Cas12i Nuclease activity (such as DNA double-strand or single-strand cleavage activity) is reduced by at least about 50% (such as a reduction of at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%) , 98%, 99%, or any of 100%).
  • the Cas12i nuclease activity can be reduced by several methods, for example, introducing mutations into one or more domains of the Cas12i nuclease: a domain interacting with PAM, a domain involved in opening a DNA double strand, RuvC domain, region interacting with nucleic acid (DNA/RNA), etc.
  • the catalytic residues of the Cas12i nuclease activity can be replaced by different amino acid residues (for example, glycine or alanine) to reduce the the nuclease activity.
  • Examples of such mutations of Cas12i1 include D647A, E894A and/or D948A.
  • Examples of such mutations of Cas12i2 include D599A, E833A, S883A, H884A, D886A, R900A and/or D1019A.
  • the engineered Cas12i nuclease or a functional derivative thereof comprises one of the following or multiple nuclease activity deletion mutations: D599A, E833A, S883A, H884A, R900A and D1019A; wherein, the amino acid position is defined as the corresponding amino acid position shown in SEQ ID NO:1.
  • the engineered Cas12i nuclease (or functional variant thereof) has increased activity compared to the reference Cas12i nuclease. In some embodiments, the engineered Cas12i nuclease (or functional variant thereof) has reduced activity compared to the reference Cas12i nuclease. In some embodiments, the activity is target DNA binding activity. In some embodiments, the activity is a site-specific nuclease activity. In some embodiments, the activity is the activity of opening double strands of DNA. In some embodiments, the activity is double-stranded DNA cleavage activity.
  • the activity is single-stranded DNA cleavage activity, including, for example, site-specific DNA cleavage activity or non-specific DNA cleavage activity.
  • the activity is single-stranded RNA cleavage activity, eg, site-specific RNA cleavage activity or non-specific RNA cleavage activity.
  • the activity is measured in vitro.
  • the activity is measured in cells, such as bacterial cells, plant cells, or eukaryotic cells.
  • the activity is measured in mammalian cells, such as rodent cells or human cells.
  • the activity is measured in human cells, such as 293T cells.
  • the activity is measured in mouse cells, eg, Hepal-6 cells.
  • the engineered Cas12i nuclease (or a functional variant thereof) has an increase of at least about 10%, 20%, 30%, 40%, 50%, 60% compared to a reference Cas12i nuclease , 70%, 80%, 90%, 95%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more in any one of the activities (as above-mentioned one one one or more activities, such as site-specific nuclease activity).
  • the engineered Cas12i nuclease (or a functional variant thereof) has at least about a 10%, 20%, 30%, 40%, 50%, 60% reduction compared to a reference Cas12i nuclease , 70%, 80%, 90%, 95%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more in any one of the activities (as above-mentioned one one or more activities, such as site-specific nuclease activity).
  • the site-specific nuclease activity of the engineered Cas12i nuclease can be measured using methods known in the art, including, for example, gel shift assays, as in the Examples provided herein The described in vitro cleavage assay based on agarose gel electrophoresis.
  • the activity is gene editing activity in the cell.
  • the cells are bacterial cells, plant cells, or eukaryotic cells.
  • the cells are mammalian cells such as rodent cells or human cells.
  • the cells are 293T cells.
  • the activity is measured in mouse cells, such as Hepal-6 cells.
  • the activity is an indel forming activity at a target genomic site in the cell, for example, site-specific cleavage and DNA repair occurs through the mechanism of non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • the activity is the activity of inserting an exogenous nucleic acid sequence at a target genomic site in the cell, for example by performing site-specificity on the target nucleic acid through the engineered Cas12i nuclease (or a functional variant thereof) Cleavage and DNA repair by homologous recombination (HR; by further introducing a repair template) mechanism.
  • the engineered Cas12i nuclease (or its functional variant) is compared with the reference Cas12i nuclease in the target genome of cells (e.g.
  • human cells such as 293T cells, or mouse Hepa1-6 cells
  • the engineered Cas12i nuclease (or functional variant thereof) is more active in cells (e.g., human cells such as 293T cells or mouse Hepa1-6 cells) than the reference Cas12i2 nuclease ( For example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) target genomic loci are increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1 fold, 1.1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold or more of gene editing (e.g. Deletion formation, or repair) activity.
  • gene editing e.g. Deletion formation, or repair
  • the engineered Cas12i nuclease (or functional variant thereof) is capable of editing a greater number (e.g., 2, 3, 4, 5, 6, 7) than the reference Cas12i nuclease , 8, 9, 10, 20, 30, 40, 50, 60, 70 or more) genomic loci, such as identifying more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70 or more) of PAM sequences.
  • the consensus PAM sequence of the engineered Cas12i nuclease (or functional variant thereof) is identical to the reference Cas12i nuclease.
  • the gene editing efficiency of engineered Cas12i nucleases (or functional variants thereof) in vitro or in cells can be determined using methods known in the art, including, for example, T7 endonuclease 1 (T7E1) assays, sequencing of target DNA (including For example, Sanger sequencing, and next-generation sequencing), detection of indels by decomposition (TIDE), or detection of indels by amplicon analysis (IDAA).
  • T7E1 T7 endonuclease 1
  • sequencing of target DNA including For example, Sanger sequencing, and next-generation sequencing
  • TIDE detection of indels by decomposition
  • IDAA detection of indels by amplicon analysis
  • targeted next-generation sequencing is used to measure the gene editing efficiency of the engineered Cas12i nuclease in cells.
  • exemplary genomic loci that can be used to determine the gene editing efficiency of the engineered Cas12i nuclease (or functional variant thereof) include, but are not limited to, CCR5, AAVS, CD34, RNF2, and EMX1.
  • the gene editing efficiency of the engineered Cas12i nuclease is that the engineered Cas12i nuclease is at least 5, 10, 15, 20, 25 , the average gene editing efficiency of 30, 35, 40, 45, 50, 55, 60, 65 or more loci (such as genomic loci in human cells).
  • the gene editing efficiency (such as indel rate) of the engineered Cas12i nuclease (or its functional variant) reaches at least 10%, 20%, 30%, 40%, 45%, 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher.
  • the engineered Cas12i nuclease (or functional variant thereof) has increased on-target specificity compared to the reference Cas12i nuclease, with a reduced off-target rate (e.g., reduced off-target recognition) sites, and/or reduced editing efficiency at one or more off-target sites), and/or increased target sequence editing efficiency.
  • the engineered Cas12i nuclease (or functional variant thereof) is reduced by at least about 5% (such as by at least about 10%, 20%, 30%) compared to the reference Cas12i nuclease , 40%, 50%, 60%, 70%, 80%, 90%, 95%, or any one of 100%) off-target rate.
  • the engineered Cas12i nuclease (or functional variant thereof) is reduced by at least one (such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more) off-target sites.
  • the engineered Cas12i nuclease (or a functional variant thereof) has the same or similar editing efficiency to the target sequence (eg, within 1.1-fold) as compared to the reference Cas12i nuclease, or The editing efficiency of the target sequence is increased (eg, at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold or more than the reference Cas12i nuclease).
  • the engineered Cas12i nuclease (or its functional variant) reduces at least about 5% off-target rate compared with the reference Cas12i nuclease, and has the same editing efficiency to the target sequence, Similar (for example, within 1.1 times), or increased.
  • the guide RNA or crRNA comprises or consists of the following from 5' to 3': direct repeat sequence, spacer sequence. In some embodiments, the guide RNA comprises or consists of the following from 5' to 3': a direct repeat sequence, a spacer sequence, a nucleotide sequence constructed in tandem of direct repeat sequences. In some embodiments, the RNA guide comprises crRNA. In some embodiments, the guide RNA does not comprise tracrRNA.
  • the crRNAs described herein include direct repeat sequences and spacer sequences.
  • the crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or a spacer sequence.
  • the crRNA includes a direct repeat sequence, a spacer sequence, and a direct repeat sequence (DR-spacer-DR), which is a typical precursor crRNA (pre-crRNA) configuration in other CRISPR systems.
  • the crRNA includes truncated direct repeat and spacer sequences, which are typical of processed or mature crRNA.
  • the CRISPR-Cas effector protein forms a complex with the RNA guide, and the spacer sequence directs the complex for sequence-specific binding to a target nucleic acid that is complementary to the spacer sequence.
  • the RNA guide comprises direct repeats. In some embodiments, the RNA guide can form a secondary structure, eg, a stem-loop structure as described herein.
  • a CRISPR system described herein comprises a plurality of RNA guides (eg, 2, 3, 4, 5, 10, 15 or more) or a plurality of nucleic acids encoding the plurality of RNA guides.
  • the CRISPR systems described herein comprise a single RNA strand or a nucleic acid encoding a single RNA strand, wherein the RNA guides are arranged in tandem.
  • the single RNA strand can comprise multiple copies of the same RNA guide, multiple copies of different RNA guides, or a combination thereof.
  • each RNA guide is specific for a different target nucleic acid.
  • the CRISPR systems described herein include RNA guides or nucleic acids encoding RNA guides.
  • the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (eg, hybridizing under appropriate conditions) to a target nucleic acid.
  • the RNA guide sequence can be modified in a manner that allows formation of the CRISPR effector complex and successful binding of the target sequence, while not allowing successful nuclease activity (i.e., no nuclease activity/causing indels).
  • modified guide sequences are referred to as "inactivated guides” or “inactivated guide sequences”.
  • These inactive guides or inactive guide sequences may be catalytically inactive or conformationally inactive with respect to nuclease activity. Inactive guide sequences are generally shorter than corresponding guide sequences that result in cleavage of active RNA.
  • an inactive guide is at least about 5%, 10%, 20%, 30%, 40%, or 50% shorter than a corresponding RNA guide having nuclease activity.
  • the inactive guide sequence of the RNA guide can be 13 to 15 nucleotides (e.g., 13, 14, or 15 nucleotides), 15 to 19 nucleotides, or 17 to 18 nucleotides in length (eg, 17 nucleotides).
  • the inactivated guide RNA is capable of hybridizing to the target sequence such that the CRISPR system is directed to the genomic locus of interest in the cell without detectable cleavage activity.
  • RNA guide sequence comprises base modifications.
  • the present invention also provides all possible variants of a nucleic acid (eg, cDNA) that can be prepared by selecting combinations based on possible codon usage. These combinations are made according to the standard triplet genetic code applied to polynucleotides encoding naturally occurring variants, and all such variants are considered specifically disclosed.
  • a nucleic acid eg, cDNA
  • the spacer sequence (or spacer, guide sequence) can be complementary to the target sequence of the target nucleic acid (such as DNA), such as at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) complementary.
  • the spacer sequence is at least 15 (e.g., at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) from the target sequence of the target nucleic acid (e.g., DNA). , 29, 30, 35, 40, 45, 50 or more) nucleotides complementary.
  • cleavage efficiency can be exploited by introducing mismatches (e.g., one or more mismatches, e.g., 1 or 2 mismatches between the spacer sequence and the target sequence, including the position of the mismatch along the spacer/target). adjust. Mismatches, such as double mismatches, are more centrally located (i.e., not at the 3' or 5' ends); cleavage efficiency is more affected. Thus, by selecting the position of the mismatch along the spacer sequence, the efficiency of cleavage can be tuned. For example, if less than 100% cleavage of the target is desired (eg, in a population of cells), 1 or 2 mismatches between the spacer sequence and the target sequence may be introduced in the spacer sequence.
  • mismatches e.g., one or more mismatches, e.g., 1 or 2 mismatches between the spacer sequence and the target sequence, including the position of the mismatch along the spacer/target.
  • the guide sequence can be of suitable length.
  • the spacer length of the RNA guide can range from about 11 to 50 (eg, about 15 to 50) nucleotides.
  • the guide sequence is between about 18 and about 35 nucleotides, including, for example, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34 or 35 nucleotides.
  • the RNA guide has a spacer length of at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides or at least 22 nucleotides.
  • the spacer is 15 to 17 nucleotides, 15 to 23 nucleotides, 16 to 22 nucleotides, 17 to 20 nucleotides, 20 to 24 nucleotides in length ( For example, 20, 21, 22, 23 or 24 nucleotides), 23 to 25 nucleotides (for example, 23, 24 or 25 nucleotides), 24 to 27 nucleotides, 27 to 30 nucleotides nucleotides, 30 to 45 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 40 or 45 nucleotides), 30 or 35 to 40 nucleotides, 41 to 45 cores nucleotides, 45 to 50 nucleotides, or longer.
  • 20 to 24 nucleotides in length For example, 20, 21, 22, 23 or 24 nucleotides), 23 to 25 nucleotides (for example, 23, 24 or 25 nucleotides), 24 to 27 nucleotides, 27 to 30 nucleotides nucleotides, 30 to 45 nu
  • the spacer of the RNA guide is 31 nucleotides in length. In some embodiments, the RNA guide has a direct repeat length of at least 21 nucleotides, or between 21 and 37 nucleotides (eg, 23, 24, 25, 30, 35, or 36 nucleotides). In some embodiments, the spacer sequence comprises or consists of about 15 to about 34 nucleotides (eg, 16, 17, 18, 19, 20, 21 or 22 nucleotides). In some embodiments, the spacer sequence is between 17 and 31 nucleotides in length. In some embodiments, the spacer sequence is between 15 and 24 nucleotides in length. In some embodiments, the direct repeat length of the RNA guide is 23 or 20 nucleotides.
  • the direct repeat sequence can guide the Cas12i protein (such as any engineered Cas12i nuclease of the present invention, its functional variant or effector protein) to bind to the guide gRNA (or crRNA) to form a CRISPR-Cas complex targeting the target sequence .
  • Cas12i protein such as any engineered Cas12i nuclease of the present invention, its functional variant or effector protein
  • Any DR that can guide the Cas12i nuclease or effector protein engineered in the application to bind to the guide gRNA (or crRNA) to form a CRISPR-Cas complex targeting the target sequence can be used in the present invention, such as US11168324 (the entire content of which The DR sequences described herein are incorporated by reference in their entirety.
  • a direct repeat can consist of two stretches of nucleotides, which can be complementary to each other, separated by an intervening nucleotide, such that the direct repeat can hybridize to form a double-stranded RNA duplex (dsRNA duplex), resulting in a stem-loop structure , in which two stretches of complementary nucleotides form a stem, and intervening nucleotides form a loop or hairpin.
  • dsRNA duplex double-stranded RNA duplex
  • the intermediate nucleotides forming a "loop” have a length of from about 6 nucleotides to about 8 nucleotides, or about 7 nucleotides.
  • the stem may comprise at least 2, at least 3, at least 4 or 5 base pairs.
  • a direct repeat may comprise two complementary stretches of nucleotides of about 4 to about 7 (eg, 4, 5, 6, 7) nucleotides in length, separated by about 5 to about 9 (such as 5, 6, 7, 8, 9) nucleotides.
  • Those skilled in the art can model known direct repeat structures.
  • Direct repeats may comprise or consist of about 13 to about 23 nucleotides, about 22 to about 40 nucleotides, or about 23 to about 38 nucleotides, or about 23 to about 36 nucleotides .
  • the direct repeat sequence includes a stem-loop structure near the 3' end (immediately adjacent to the spacer sequence). In some embodiments, the direct repeat sequence includes a stem-loop near the 3' end, wherein the stem is 5 nucleotides in length. In some embodiments, the direct repeat sequence includes a stem-loop near the 3' end, wherein the stem is 5 nucleotides in length and the loop is 7 nucleotides in length. In some embodiments, the direct repeat sequence includes a stem-loop near the 3' end, wherein the stem is 5 nucleotides in length and the loop is 6, 7 or 8 nucleotides in length.
  • the direct repeat sequence includes near the 3' end the sequence 5'-CCGUCNNNNNNNUGACGG-3' (SEQ ID NO: 68), where N refers to any nucleobase. In some embodiments, the direct repeat sequence includes near the 3' end the sequence 5'-GUGCCNNNNNNNUGGCAC-3' (SEQ ID NO: 69), where N refers to any nucleobase.
  • the direct repeat sequence includes the sequence 5'-GUGUCN 5-6 UGACAX 1 -3' (SEQ ID NO: 70 or 71) near the 3' end, wherein N 5-6 refers to any 5 or 6 A contiguous sequence of nucleobases, and X 1 refers to C or T or U.
  • the direct repeat sequence includes the sequence 5'-UCX 3 UX 5 X 6 X 7 UUGACGG-3' (SEQ ID NO: 72) near the 3' end, wherein X 3 refers to C or T or U, X5 means A or T or U, X6 means A or C or G, and X7 means A or G.
  • the direct repeat sequence includes the sequence 5'-CCX 3 X 4 X 5 CX 7 UUGGCAC-3' (SEQ ID NO: 73) near the 3' end, wherein X 3 refers to C or T or U, X4 means A or T or U, X5 means C or T or U, and X7 means A or G.
  • the nucleotides encoding the direct repeat sequence comprise or consist of at least about 80%, such as at least about 85%, identical to SEQ ID NO: 59 (AGAAATCCGTCTTTCATTGACGG) or SEQ ID NO: 79 (GTTGCAAAACCCAAGAAATCCGTCTTTCATTGACGG) , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical nucleotide sequences.
  • the nucleotides encoding the direct repeat sequence comprise at least 21 (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36) nucleotides.
  • “Stem-loop structure” refers to a nucleic acid having a secondary structure comprising a region of nucleotides known or predicted to form a double strand (stem portion) flanked on one side by the main Regions (loop portions) that are single-stranded nucleotides are connected.
  • the terms “hairpin” and “fold-back” structures are also used herein to refer to stem-loop structures. Such structures are well known in the art, and these terms are used consistently with their art-known meanings.
  • stem-loop structures do not require precise base pairing.
  • the stem may include one or more base mismatches.
  • base pairing can be exact, ie not include any mismatches.
  • the stem of the direct repeat consists of 5 complementary nucleobases that hybridize to each other and the loop is 6, 7 or 9 nucleotides in length.
  • the sequence encoding the direct repeat comprises or consists of the following: the sequence shown in SEQ ID NO:79. In some embodiments, the sequence encoding the direct repeat comprises or consists of the following: the truncated nucleic acid with the initial three 5' nucleotides of the SEQ ID NO:79 nucleic acid sequence. In some embodiments, the sequence encoding the direct repeat comprises or consists of the following: the truncated nucleic acid with the initial four 5' nucleotides of the SEQ ID NO:79 nucleic acid sequence.
  • the sequence encoding the direct repeat comprises or consists of the following: the truncated nucleic acid with the initial five 5' nucleotides of the SEQ ID NO:79 nucleic acid sequence. In some embodiments, the sequence encoding the direct repeat comprises or consists of the following: the truncated nucleic acid with the initial six 5' nucleotides of the SEQ ID NO:79 nucleic acid sequence. In some embodiments, the sequence encoding the direct repeat comprises or consists of the following: the truncated nucleic acid with the initial seven 5' nucleotides of the SEQ ID NO:79 nucleic acid sequence.
  • sequence encoding the direct repeat comprises or consists of the following: the truncated nucleic acid with the initial eight 5' nucleotides of the SEQ ID NO:79 nucleic acid sequence. In some embodiments, the sequence encoding the direct repeat comprises or consists of the following: the sequence shown in SEQ ID NO:59.
  • the direct repeat is a "functional variant" of the RNA sequence encoded by SEQ ID NO: 59 or 79, such as a “functionally truncated version", a “functionally extended version”, or a “functionally substituted version”. ", such as a part of SEQ ID NO:79 (truncated version), still has DR function.
  • a DR "functional variant” is a 5' and/or 3' extension (functionally extended version) or truncation (functionally truncated version) of a reference DR (such as a parental DR), and/or a reference DR sequence
  • a reference DR such as a parental DR
  • DR sequence After inserting, deleting, and/or replacing (functional replacement version) one or more nucleotides, still have at least 20% of the reference DR (such as at least about any 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher) function of the DR sequence, that is, the function of mediating the combination of the Cas12i protein and the corresponding crRNA.
  • DR functional variants generally retain the stem-loop-like secondary structure or part thereof available for Cas12i protein binding.
  • the DR or its functional variant comprises a stem-loop-like secondary structure or part thereof available for Cas12i protein binding. In some embodiments, the DR or its functional variant comprises at least two (such as 2, 3, 4, 5 or more) stem-loop-like secondary structures or parts thereof available for Cas12i protein binding.
  • pegRNA Prime editing guide RNA
  • PEgRNAs (relative to standard guide RNAs) are altered to include an extension that provides a DNA synthesis template sequence encoding a single-stranded DNA flap that is homologous to the strand to be edited targeting the endogenous DNA sequence but contains the desired one or multiple nucleotide changes and are incorporated into the target DNA molecule after being synthesized by a polymerase (eg, reverse transcriptase).
  • a polymerase eg, reverse transcriptase
  • the extended guide RNA comprises (a) a guide RNA and (b) an RNA extension at the 5' or 3' end of the guide RNA or in an intramolecular position of the guide RNA.
  • the intramolecular positioning of the extension does not disrupt the function of the protospacer.
  • the RNA extension may comprise (i) a reverse transcription template sequence comprising the desired nucleotide changes, (ii) a reverse transcription primer binding site, and (iii) an optional linker sequence.
  • the reverse transcription template sequence may encode a single-stranded DNA flap complementary to an endogenous DNA sequence adjacent to the nicking site, wherein the single-stranded DNA flap contains the desired nucleotide change.
  • the ssDNA flap displaces endogenous ssDNA at the nicking site.
  • the desired nucleotide change incorporated into the target DNA can be a single nucleotide change (e.g., a transition or transversion), an insertion of one or more nucleotides, or one or more nuclear Deletion of nucleotides.
  • the desired nucleotide change may be a single nucleotide substitution (eg, transition or transversion), deletion or insertion.
  • the desired nucleotide change may be (1) G to T substitution, (2) G to A substitution, (3) G to C substitution, (4) T to G substitution, (5) T to A substitution, (6) T to C substitution, (7) C to G substitution, (8) C to T substitution, (9) C to A substitution, (10) A to T substitution, (11) A to G substitution, or ( 12) A to C substitution.
  • the engineered Cas12i nuclease (or its functional variant) of the present invention can recognize the protospacer adjacent motif (PAM).
  • the target nucleic acid comprises a PAM.
  • the PAM is located 5' to a sequence in the target nucleic acid that is complementary to the targeting sequence of the guide RNA.
  • the PAM comprises or consists of the nucleic acid sequence 5'-TTN-3', 5'-TTH-3', 5'-TTY-3' or 5'-TTC-3'.
  • the PAM comprises or consists of the nucleic acid sequence 5'-TTTN-3'.
  • the PAM comprises or consists of the nucleic acid sequence 5'-TTN-3'.
  • the PAM applicable to the engineered Cas12i nuclease (or its functional variant) of the present invention includes the nucleic acid sequence 5'-TTTA-3', 5'-CTTA-3', 5'- GTTA-3', 5'-ATTA-3', 5'-TTTC-3', 5'-CTTC-3', 5'-GTTC-3', 5'-ATTC-3', 5'-TTTG- 3', 5'-CTTG-3', 5'-GTTG-3', 5'-ATTG-3', 5'-TTTT-3', 5'-CTTT-3', 5'-GTTT-3' , 5'-ATTT-3'.
  • the application provides engineered Cas12i (such as Cas12i2) effector protein, which comprises any engineered Cas12i nuclease of the present invention or its functional variant (such as CasXX shown in SEQ ID NO: 8), which has improved activity , such as target binding, double-strand cleavage activity, nickase activity, and/or gene editing activity.
  • engineered Cas12i such as Cas12i2
  • effector protein which comprises any engineered Cas12i nuclease of the present invention or its functional variant (such as CasXX shown in SEQ ID NO: 8), which has improved activity , such as target binding, double-strand cleavage activity, nickase activity, and/or gene editing activity.
  • an engineered Cas12i effector protein (e.g., a Cas12i nuclease, a Cas12i nickase, a Cas12i fusion effector protein, or a split Cas12i effector protein) comprising an engineered Cas12i nucleic acid as described herein is provided Any of the enzymes or their functional derivatives (such as dCas12i).
  • the engineered Cas12i effector protein comprises, consists essentially of, or consists of any of the engineered Cas12i nucleases described herein or a functional variant thereof.
  • an engineered Cas12i effector protein based on any of the engineered Cas12i2 nucleases described herein or functional variants thereof (e.g., CasXX shown in SEQ ID NO: 8).
  • the engineered Cas12i effector protein has enzymatic activity (eg, DNA double-strand cleavage activity).
  • the engineered Cas12i effector protein has nuclease activity that cleaves both strands of a target duplex nucleic acid (eg, duplex DNA).
  • the engineered Cas12i effector protein has nickase activity, ie, cleaves a single strand of a target duplex nucleic acid (eg, duplex DNA).
  • the engineered Cas12i effector protein comprises an enzyme-inactive mutant of the engineered Cas12i nuclease.
  • the application also provides any split-type Cas12i effector protein based on the engineered Cas12i nuclease described herein or a functional variant thereof (such as CasXX shown in SEQ ID NO: 8).
  • Split Cas12i effectors may be advantageous for delivery.
  • the engineered Cas12i effector protein is split into two parts of the enzyme that can be reconstituted together to provide a substantially functioning Cas12i effector protein.
  • Cas effector proteins can be provided using known methods, for example, fragmented forms of the Cas12 and Cas9 proteins have been described in, for example, WO2016/112242, WO2016/205749 and PCT/CN 2020/111057, which are incorporated herein by reference in their entirety.
  • a split-type Cas12i effector protein comprising a first polypeptide comprising any of the engineered Cas12i nucleases described herein or a functional derivative thereof and a second polypeptide
  • the N-terminal portion of the species, the second polypeptide comprises the C-terminal portion of any one of the engineered Cas12i nuclease or its functional derivatives, wherein the first polypeptide and the second polypeptide are capable of comprising
  • the guide RNAs of the guide sequence associate with each other in the presence of each other to form a CRISPR complex that specifically binds to a target nucleic acid comprising a target sequence complementary to the guide sequence.
  • the first polypeptide and the second polypeptide each comprise a dimerization domain. In some embodiments, the first dimerization domain and the second dimerization domain associate with each other in the presence of an inducing agent (eg, rapamycin). In some embodiments, the first and second polypeptides do not comprise a dimerization domain. In some embodiments, the segmented Cas12i effector protein is autoinducible.
  • the engineered Cas12i effector protein of the present invention can be split in a manner that does not affect the catalytic domain.
  • Cas12i effector proteins can act as nucleases (including nickases) or can be inactivated enzymes, which are essentially RNA-guided DNA-binding proteins with little or no catalytic activity (e.g. due to mutations in their catalytic domain) .
  • the nuclease lobe and the ⁇ -helical lobe of the engineered Cas12i effector protein are expressed as separate polypeptides.
  • the RNA guide sequence recruits them into a complex that recapitulates the activity of the full-length Cas12i nuclease and catalyzes site-specific DNA cutting.
  • modified RNA guide sequences can be used to eliminate the activity of partitioned enzymes by preventing dimerization, thereby allowing the development of an inducible dimerization system.
  • split-type enzymes are described, for example, in Wright, Addison V., et al. "Rational design of a split-Cas9 enzyme complex," Proc. Nat'l. Acad. Sci., 112.10 (2015): 2984-2989, It is incorporated herein by reference in its entirety.
  • the split-type Cas12i effector protein portion described herein can be designed as a reference engineered Cas12i effector protein (e.g., a full-length engineered Cas12i nuclease) at a split position (i.e., split) into two halves, the Position is the point where the N-terminal portion of the reference Cas12i effector protein is separated from the C-terminal portion.
  • the N-terminal portion comprises amino acid residues 1 to X of the reference Cas12i effector protein
  • the C-terminal portion comprises amino acid residues X+1 to the C-terminus of the reference Cas12i effector protein .
  • the numbering is consecutive, but this is not required, as it is also contemplated that the amino acids (or the nucleotides encoding them) can be anywhere from split ends and/or mutations (e.g., insertions, deletions, and substitutions).
  • One is trimmed from the internal region of the polypeptide chain, provided that the reconstituted engineered Cas12i effector retains sufficient DNA binding activity (if desired), DNA nickase or cleavage activity, e.g., with the reference Cas12i effector At least about 40% (eg, at least about 50%, 60%, 70%, 80%, 90%, 95% or more) activity compared to a protein.
  • Cutpoints can be designed in silico and cloned into constructs. During this process, mutations can be introduced into segmented Cas12i effectors and non-functional domains can be removed.
  • the two parts or fragments (i.e., N-terminal and C-terminal fragments) of the split-type Cas12i effector protein can form a complete Cas12i effector protein comprising, for example, at least about 70% of the complete Cas12i effector protein sequence (eg, at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or more).
  • the segmented Cas12i effector proteins may each comprise one or more dimerization domains.
  • the first polypeptide comprises a first dimerization domain fused to a first split-type Cas12i effector protein portion
  • the second polypeptide comprises a first dimerization domain fused to a second split-type Cas12i effector protein portion.
  • Second dimerization domain can be fused to the segmented Cas12i effector protein portion by a peptide linker (eg, a flexible peptide linker such as a GS linker) or a chemical bond.
  • the dimerization domain is fused to the N-terminus of the segmented Cas12i effector protein portion.
  • the dimerization domain is fused to the C-terminus of the segmented Cas12i effector protein portion.
  • the segmented Cas12i effector protein does not comprise any dimerization domains.
  • the dimerization domain facilitates the association of two segmented Cas12i effector protein moieties.
  • the segmented Cas12i effector portion is induced by an inducer to associate or dimerize into a functional Cas12i effector.
  • the segmented Cas12i effector protein comprises an inducible dimerization domain.
  • the dimerization domain is not an inducible dimerization domain, ie, the dimerization domain dimerizes in the absence of an inducing agent.
  • An inducing agent may be an inducing energy source or an inducing molecule other than a guide RNA (eg, crRNA).
  • the inducer partially remodels the two segmented Cas12i effectors into a functional Cas12i effector through the induced dimerization of the dimerization domain.
  • the inducing agent brings together the two segmented Cas12i effector protein moieties by inducing association of the inducible dimerization domain.
  • the two split Cas12i effector protein portions do not associate with each other to reconstitute into a functional Cas12i effector protein in the absence of an inducing agent.
  • two separate Cas12i effector portions can associate with each other in the presence of a guide RNA (eg, crRNA) to remodel into a functional Cas12i effector.
  • the inducer of the present application may be heat, ultrasound, electromagnetic energy or chemical compounds.
  • the inducing agent is an antibiotic, small molecule, hormone, hormone derivative, steroid, or steroid derivative.
  • the inducer is abscisic acid (ABA), doxycycline (DOX), cumate, rapamycin, 4-hydroxytamoxifen (4OHT), Estrogen or ecdysone.
  • the segmented Cas12i effector system is an inducer-controlled system selected from the group consisting of antibiotic-based induction systems, electromagnetic energy-based induction systems, small molecule-based induction systems, nuclear receptor-based induction systems Induced systems and hormone-based induced systems.
  • the segmented Cas12i effector system is an inducer-controlled system selected from the group consisting of tetracycline (Tet)/DOX inducible system, light inducible system, ABA inducible system, cumate The repressor/operator system, the 4OHT/estrogen inducible system, the ecdysone-based inducible system and the FKBP12/FRAP (FKBP12-rapamycin complex) inducible system.
  • inducers are also discussed herein and in PCT/US2013/051418, which is hereby incorporated by reference in its entirety.
  • pairs of split-type Cas12i effector proteins are separated and inactive until dimerization of the dimerization domain (e.g., FRB and FKBP) is induced, which results in Reassembly of functional Cas12i effector protein nucleases.
  • the first split-type Cas12i effector protein comprising the first half of the inducible dimer (eg, FRB) is delivered separately, and/or in a separate manner from the first half comprising the inducible dimer (eg, FKBP). The position where the second split-type Cas12i effector separates in the second half.
  • FKBP-based induction systems that can be used in the inducer-controlled split Cas12i effector systems described herein include, but are not limited to: FKBP that dimerizes with calcineurin (CNA) in the presence of FK506 ; FKBP that dimerizes with CyP-Fas in the presence of FKCsA; FKBP that dimerizes with FRB in the presence of rapamycin; GyrB that dimerizes with GryB in the presence of coumarin; GAI that dimerizes with GID1 in the presence of mycin; or Snap-tag that dimerizes with HaloTag in the presence of HaXS.
  • CNA calcineurin
  • FKBPs homodimerize (ie, one FKBP dimerizes with another FKBP) in the presence of FK1012.
  • the dimerization domain is FKBP and the inducer is FK1012. In some embodiments, the dimerization domain is GryB and the inducer is coumarycin. In some embodiments, the dimerization domain is ABA and the inducing agent is gibberellin.
  • the segmented Cas12i effector portion can be autoinduced (ie, autoactivated or autoinduced) in the absence of an inducing agent to associate/dimerize into a functional Cas12i effector.
  • the auto-induction of the segmented Cas12i effector portion may be mediated by binding to a guide RNA such as crRNA.
  • the first and second polypeptides do not comprise a dimerization domain. In some embodiments, the first and second polypeptides comprise a dimerization domain.
  • the reconstituted Cas12i effector protein of the split-type Cas12i effector system described herein has an editing efficiency of at least about 60% ( An editing efficiency such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more).
  • the reconstituted Cas12i effector protein of the inducer-controlled split-type Cas12i effector system described herein in the absence of an inducer (i.e., due to auto-induction), has a higher editing efficiency relative to a reference Cas12i effector protein. Having an editing efficiency of no more than about 50%, such as no more than any of about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less efficiency.
  • the application also provides engineered Cas12i effector proteins comprising additional protein domains and/or components, such as linkers, nuclear localization/export sequences, functional domains and/or reporter proteins.
  • the engineered Cas12i effector protein comprises one or more heterologous protein domains (e.g., about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more structural domains) and the protein complex of the nucleic acid targeting structural domain or functional derivative thereof of any engineered Cas12i nuclease of the present invention.
  • the engineered Cas12i effector protein comprises one or more heterologous proteins fused with the engineered Cas12i nuclease or a functional variant thereof (such as CasXX shown in SEQ ID NO: 8) Fusion proteins of domains (eg, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains).
  • the engineered Cas12i effector protein of the present application may comprise (for example, through a fusion protein, such as through one or more peptide linkers, such as a GS peptide linker, etc.) one or more functional domains or association (for example, by co-expression of multiple proteins) on it.
  • the one or more functional domains are enzymatic domains.
  • RNA methylase activity DNA and/or RNA methylase activity
  • nucleotide deaminase activity such as adenosine deaminase activity, cytidine deaminase activity
  • demethylase activity transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity (e.g., double-strand endonuclease activity, nickase activity), nucleic acid binding activity, and switch activity (e.g., photoinduced or chemically induced).
  • the one or more functional domains are transcriptional activation domains (ie, transactivation domains) or repressor domains. In some embodiments, the one or more functional domains are histone modification domains. In some embodiments, the one or more functional domains are transposase domains, HR (homologous recombination) machinery domains, recombinase domains, and/or integrase domains. In some embodiments, the functional domain is Krüppe1-associated box (KRAB), VP64, VP16, Fok1, P65, HSF1, MyoD1, Biotin-APEX, APOBEC1, AID, PmCDA1, Tad1, and M-MLV reverse transcriptase .
  • KRAB Krüppe1-associated box
  • the functional domain is selected from the group consisting of translation initiation domain, transcriptional repression domain, transactivation domain, epigenetic modification domain, nucleobase editing domain (e.g., CBE or ABE domain), reverse transcriptase domain, reporter domain (eg, fluorescent domain) and nuclease domain.
  • the functional domain has the activity of modifying target DNA or target DNA-related protein selected from nuclease activity (for example, HNH nuclease, RuvC nuclease, Trex1 nuclease, Trex2 nuclease) , methylation activity, demethylation activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity , transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphate Enzyme activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity
  • the positioning of one or more functional domains in the engineered Cas12i effector protein allows correct spatial orientation of the functional domains to affect a target with the conferred functional effect.
  • the functional domain is a transcriptional activator (e.g., VP16, VP64, or p65)
  • the transcriptional activator is placed in a spatial orientation that enables it to affect the transcription of the target.
  • a transcriptional repressor is positioned to affect the transcription of a target, and a nuclease (eg, Fok1 ) is positioned to cleave or partially cleave the target.
  • the functional domain is located at the N-terminus of the engineered Cas12i effector protein.
  • the functional domain is located at the C-terminus of the engineered Cas12i effector protein.
  • the engineered Cas12i effector protein comprises a first functional domain at the N-terminus and a second functional domain at the C-terminus.
  • the engineered Cas12i effector protein comprises a catalytically inactive mutant (dCas12i) of any of the engineered Cas12i nucleases described herein fused to one or more functional domains.
  • the engineered Cas12i effector protein is a transcriptional activator.
  • the engineered Cas12i effector protein comprises an enzyme-inactive variant of any of the engineered Cas12i nucleases described herein fused to a transactivation domain.
  • the transactivation domain is selected from the group consisting of VP64, p65, HSF1, VP16, MyoD1, HSF1, RTA, SET7/9, and combinations thereof.
  • the transactivation domain comprises VP64, p65, and HSF1.
  • the engineered Cas12i effector protein comprises two split Cas12i effector polypeptides, each fused to a transactivation domain.
  • the engineered Cas12i effector protein is a transcriptional repressor.
  • the engineered Cas12i effector protein comprises an enzyme-inactive variant of any of the engineered Cas12i nucleases described herein fused to a transcriptional repression domain.
  • the transcriptional repressor domain is selected from the group consisting of Krüppel-associated box (KRAB), EnR, NuE, NcoR, SID, SID4X, and combinations thereof.
  • the engineered Cas12i effector protein comprises two split Cas12i effector polypeptides, each fused to a transcriptional repression domain.
  • the engineered Cas12i effector protein is a base editor, such as a cytosine editor or an adenosine editor.
  • the engineered Cas12i effector protein comprises an enzyme-inactive variant of any of any of the engineered Cas12i nucleases described herein fused to a nucleobase editing domain
  • the nucleobase Editing domains are such as cytosine base editing (CBE) domains or adenosine base editing (ABE) domains.
  • the nucleobase editing domain is a DNA editing domain.
  • the nucleobase editing domain has deaminase activity.
  • the nucleobase editing domain is a cytosine deaminase domain. In some embodiments, the nucleobase editing domain is an adenosine deaminase domain.
  • Exemplary base editors based on Cas nucleases are described, eg, in WO2018/165629A1 and WO2019/226953A1, which are incorporated herein by reference in their entirety.
  • Exemplary CBE domains include, but are not limited to, activation-induced cytidine deaminase or AID (e.g., hAID), apolipoprotein B mRNA editing complex, or APOBEC (e.g., rat APOBEC1, hAPOBEC3A/B/C/D/E /F/G) and PmCDA1.
  • AID e.g., hAID
  • APOBEC e.g., rat APOBEC1, hAPOBEC3A/B/C/D/E /F/G
  • PmCDA1 e.g., PmCDA1.
  • Exemplary ABE domains include, but are not limited to: TadA, ABE8, and variants thereof (see, e.g., Gaudelli et al., 2017, Nature 551:464-471; and Richter et al., 2020, Nature Biotechnology 38:883-891) .
  • the functional domain is an APOBEC1 domain, eg, a rat APOBEC1 domain.
  • the functional domain is a TadA domain, such as an E. coli TadA domain.
  • the engineered Cas12i effector protein further comprises one or more nuclear localization sequences.
  • the term "adenosine deaminase” or “adenosine deaminase protein” refers to a protein, polypeptide, or one or more functional domains of a protein or polypeptide that is capable of catalyzing the conversion of adenine (or molecular adenine moiety) to hypoxanthine (or the hypoxanthine moiety of the molecule) by hydrolytic deamination as shown below.
  • the adenine-containing molecule is adenosine (A) and the hypoxanthine-containing molecule is inosine (I).
  • the adenine-containing molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • Adenosine deaminases that can be used in conjunction with enzyme-inactive variants of any of the engineered Cas12i nucleases of the invention include, but are not limited to, members of the enzyme family known as RNA-acting adenosine deaminases (ADAR) , a member of the enzyme family known as tRNA-acting adenosine deaminase (ADAT), and other family members containing an adenosine deaminase domain (ADAD).
  • ADAR RNA-acting adenosine deaminases
  • ADAT tRNA-acting adenosine deaminase
  • ADAD adenosine deaminase domain
  • ADAR can perform adenosine-to-inosine editing responses to RNA/DNA and RNA/RNA duplexes.
  • the adenosine deaminase can be modified to increase its ability to edit DNA in the RNA/DNA heteroduplex of the RNA duplex.
  • the adenosine deaminase is derived from one or more metazoan species including, but not limited to, mammals, birds, frogs, squid, fish, flies, and worms. In some embodiments, the adenosine deaminase is human, squid, or Drosophila adenosine deaminase.
  • the adenosine deaminase is a human ADAR, including hADAR1, hADAR2, hADAR3. In some embodiments, the adenosine deaminase is a Caenorhabditis elegans ADAR protein, including ADR-1 and ADR-2. In some embodiments, the adenosine deaminase is a Drosophila ADAR protein, including dAdar. In some embodiments, the adenosine deaminase is a squid (Loligo pealeii) ADAR protein, including sqADAR2a and sqADAR2b.
  • the adenosine deaminase is the human ADAT protein. In some embodiments, the adenosine deaminase is the Drosophila ADAT protein. In some embodiments, the adenosine deaminase is a human ADAD protein, including TENR (hADAD1) and TENRL (hADAD2). In some embodiments, the adenosine deaminase is TadA8e.
  • the adenosine deaminase is a TadA protein, such as E. coli TadA. See Kim et al., Biochemistry 45:6407-6416 (2006); Wolf et al., EMBO J. 21:3841-3851 (2002).
  • the adenosine deaminase is mouse ADA. See Grunebaum et al., Curr. Opin. Allergy Clin. Immunol. 13:630-638 (2013).
  • the adenosine deaminase is human ADAT2. See Fukui et al., J. Nucleic Acids 2010:260512 (2010).
  • the deaminase e.g., adenosine or cytidine deaminase
  • the deaminase is one or more of those described in: Cox et al., Science. 2017 Nov 24; 358( 6366):1019-1027; Komore et al., Nature. 2016 May 19;533(7603):420-4; and Gaudelli et al., Nature. 2017 Nov 23;551(7681):464- 471.
  • the adenosine deaminase protein comprises one or more deaminase domains.
  • the deaminase domain serves to recognize and convert one or more target adenosine (A) residues contained in double-stranded nucleic acid substrates to inosine (I) residues .
  • the deaminase is cytidine deaminase.
  • the term "cytidine deaminase” or “cytidine deaminase protein” refers to a protein, polypeptide, or one or more functional domains of a protein or polypeptide that is capable of catalyzing the conversion of cytosine (or The hydrolytic deamination reaction that converts the pyrimidine portion) to uracil (or the uracil portion of the molecule).
  • the cytosine-containing molecule is cytidine (C) and the uracil-containing molecule is uridine (U).
  • the cytosine-containing molecule may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • Cytidine deaminases that can be used in conjunction with enzyme-inactive variants of any of the engineered Cas12i nucleases of the invention include, but are not limited to, deaminases of the family known as the apolipoprotein B mRNA editing complex (APOBEC) A member of the enzyme family, activation-induced deaminase (AID), or cytidine deaminase 1 (CDA1).
  • APOBEC apolipoprotein B mRNA editing complex
  • AID activation-induced deaminase
  • CDA1 cytidine deaminase 1
  • APOBEC1 deaminase APOBEC2 deaminase
  • APOBEC3A deaminase APOBEC3B deaminase
  • APOBEC3C deaminase APOBEC3D deaminase
  • APOBEC3E deaminase APOBEC3F deaminase
  • APOBEC3G deaminase APOBEC3H deaminase or a deaminase in APOBEC4 deaminase.
  • the cytidine deaminase is capable of targeting cytosine in a single strand of DNA. In some embodiments, the cytidine deaminase can edit on a single strand present on the outside of the binding component. In some embodiments, the cytidine deaminase can edit at a localized vesicle, eg, a localized vesicle formed by a target editing site but a mismatch in the guide sequence.
  • the cytidine deaminase may contain mutations that facilitate focusing activity, such as described in Kim et al., Nature Biotechnology (2017) 35(4):371-377 (doi: 10.1038/nbt.3803 of those.
  • the cytidine deaminase is derived from one or more metazoan species including, but not limited to, mammals, birds, frogs, squid, fish, flies, and worms. In some embodiments, the cytidine deaminase is human, primate, bovine, dog, rat, or mouse cytidine deaminase.
  • the cytidine deaminase is human APOBEC, including hAPOBEC1 or hAPOBEC3. In some embodiments, the cytidine deaminase is human AID.
  • the cytidine deaminase protein comprises one or more deaminase domains.
  • the deaminase domain serves to recognize and convert one or more target cytosine (C) residues contained in the single-stranded vesicle of the RNA duplex to uracil (U) Residues.
  • the engineered Cas12i effector protein is a master editor. Cas9-based master editors are described, e.g., in A. Anzalone et al., Nature, 2019, 576(7785):149-157, which is hereby incorporated by reference in its entirety.
  • the engineered Cas12i effector protein comprises a nickase variant of any one of the engineered Cas12i nucleases described herein fused to a reverse transcriptase domain.
  • the functional domain is a reverse transcriptase domain.
  • the reverse transcriptase domain is an M-MLV reverse transcriptase or a variant thereof, eg, an M-MLV reverse transcriptase having one or more mutations of D200N, T306K, W313F, T330P, and L603W.
  • an engineered CRISPR/Cas12i system comprising the master editor is provided.
  • the engineered CRISPR/Cas12i system further comprises a second Cas12i nickase, for example based on the same engineered Cas12i nuclease as the primary editor.
  • the engineered CRISPR/Cas12i system comprises a guide editing guide RNA (pegRNA) comprising a primer binding site and a reverse transcriptase (RT) template sequence.
  • pegRNA guide editing guide RNA
  • the application provides one or more (for example, 1, 2, 3, 4, 5, 1, 2, 3, 4, 5, 6 or more) segmented Cas12i effector system of functional domains.
  • the functional domain may be provided as part of the first and/or second segmented Cas12i effector protein, as a fusion within the construct.
  • the functional domain is usually fused to other parts of the split-type Cas12i effector protein (eg, split-type Cas12i effector protein part) through a peptide linker (such as a GS linker).
  • a peptide linker such as a GS linker
  • the engineered Cas12i effector protein comprises one or more nuclear localization sequences (NLS) and/or one or more nuclear export sequences (NES).
  • NLS sequences include, for example, PKKKRKVPG (SEQ ID NO: 66) and ASPKKKRKV (SEQ ID NO: 67).
  • NLS and/or NES can be operably linked to the N-terminal and/or C-terminal of the engineered Cas12i effector protein or a polypeptide chain in the engineered Cas12i effector protein.
  • NLS and/or NES can be connected to the N-terminal and/or C-terminal of any engineered Cas12i nuclease or functional variant thereof of the present invention.
  • the engineered Cas12i effector protein can encode additional components, such as a reporter protein.
  • the engineered Cas12i effector protein comprises a fluorescent protein, such as GFP.
  • GFP fluorescent protein
  • the engineered Cas12i effector protein is an inducible segmentable Cas12i effector system that can be used to image genomic loci.
  • an engineered Cas12i effector protein is provided, wherein the effector protein is capable of inducing double-strand breaks or single-strand breaks in DNA molecules.
  • an engineered Cas12i effector protein wherein the functional derivative of the engineered Cas12i nuclease is an enzyme inactive mutant, for example containing D599A, E833A, S883A, H884A, D886A, R900A and/or the Cas12i2 nuclease inactivation mutant of D1019A (the amino acid position is defined as the corresponding amino acid position shown in SEQ ID NO: 1), and the Cas12i1 nuclease inactivation mutant containing D647A, E894A and/or D948A (The amino acid positions are defined as the corresponding amino acid positions shown in SEQ ID NO: 13).
  • Known enzyme inactivation mutants of Cas12i2 nuclease such as any enzyme inactivation mutation of Cas12i2 nuclease described in US10808245B2 and Huang X.et al., Nature Communications, 11, Article number: 5241 (2020) can Combined with the mutations in the present application to provide functional derivatives of engineered Cas12i nucleases and their corresponding effector proteins.
  • an engineered CRISPR-Cas12i system comprising: (a) any engineered Cas12i effector protein described in the present application (such as engineered Cas12i nuclease or a functional variant thereof, For example, CasXX shown in SEQ ID NO: 8); and (b) a guide RNA comprising a guide sequence complementary to the target sequence, or one or more nucleic acids encoding the guide RNA;
  • the engineered Cas12i effector protein and the guide RNA can form a CRISPR complex, and the CRISPR complex specifically binds to a target nucleic acid comprising the target sequence and induces modification (such as double-stranded or single-stranded) of the target nucleic acid strand cleavage, base editing, etc.).
  • modification covers cleavage, base editing, replacement, repair, etc. by nucleases at target sites on double-stranded or single-stranded nucleic acids.
  • the engineered CRISPR-Cas12i system comprises: (a) any one of the engineered Cas12i effector proteins described herein (for example, any of the engineered Cas12i nucleases or functional variants thereof entity, or a nicking enzyme based on the engineered Cas12i nuclease or functional variant thereof, split-type Cas12i, transcriptional repressor, transcriptional activator, base editor, or master editor); and (b) comprising and target sequence A guide RNA of a complementary guide sequence, or one or more nucleic acids encoding the guide RNA; wherein the engineered Cas12i effector protein and the guide RNA can form a CRISPR complex that specifically binds Target nucleic acid comprising the target sequence and induce modification of the target nucleic acid (such as double-stranded or single-stranded cleavage, base editing, etc.).
  • any one of the engineered Cas12i effector proteins described herein for example, any of the engineered Cas12i nu
  • the engineered CRISPR-Cas12i system comprises an engineered Cas12i effector protein (such as an engineered Cas12i nuclease or a functional variant thereof, such as CasXX shown in SEQ ID NO: 8) and/or Or one or more nucleic acids of said guide RNA.
  • the engineered CRISPR-Cas12i system comprises an array of precursor guide RNAs that can be processed into multiple crRNAs, eg, by the engineered Cas12i effector protein.
  • the engineered CRISPR-Cas12i system comprises one or more vectors encoding the engineered Cas12i effector protein and/or the guide RNA.
  • the engineered CRISPR-Cas12i system comprises a ribonucleoprotein (RNP) complex comprising the engineered Cas12i effector protein bound to the guide RNA.
  • RNP ribonucleoprotein
  • the engineered CRISPR-Cas12i system of the present application can comprise any suitable guide RNA.
  • a guide RNA may comprise a guide sequence capable of hybridizing to a target sequence in a target nucleic acid of interest, such as a genomic site of interest in a cell.
  • the gRNA comprises a CRISPR RNA (crRNA) sequence comprising the guide sequence.
  • the crRNAs described herein include direct repeat sequences and spacer sequences.
  • the crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or a spacer sequence.
  • the crRNA includes a direct repeat sequence, a spacer sequence, and a direct repeat sequence (DR-spacer sequence-DR), which is typical of the configuration of a precursor crRNA (pre-crRNA).
  • the crRNA includes truncated direct repeat and spacer sequences, which are typical features of processed or mature crRNA.
  • the CRISPR-Cas12i effector protein forms a complex with an RNA guide sequence, and the spacer sequence directs the complex to sequence-specific binding to a target nucleic acid that is associated with the spacer
  • the sequences are complementary (eg, at least 70% complementary).
  • the guide RNA is a crRNA comprising a guide sequence.
  • the engineered CRISPR-Cas12i system comprises an array of precursor guide RNAs encoding multiple crRNAs.
  • the Cas12i effector protein cleaves the array of precursor guide RNAs to generate a plurality of crRNAs.
  • the engineered CRISPR-Cas12i system comprises an array of precursor guide RNAs encoding multiple crRNAs, wherein each crRNA comprises a different guide sequence.
  • constructs, vectors, and expression systems encoding any of the engineered Cas12i effector proteins described herein (eg, engineered Cas12i nucleases or functional variants thereof).
  • the construct, vector or expression system further comprises one or more gRNA or crRNA arrays.
  • a “vector” is a composition of matter comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Many vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites and one or more selectable markers.
  • the term “vector” should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • the vector is a viral vector.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof.
  • the vector is a phage vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), among other handbooks of virology and molecular biology.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • Recombinant virus can then be isolated and delivered to the engineered mammalian cells in vitro or ex vivo.
  • Many retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • lentiviral vectors are used.
  • self-inactivating lentiviral vectors are used.
  • the vector is an adeno-associated virus (AAV) vector, such as AAV2, AAV8, or AAV9, which can be administered in a single dose containing at least 1 x 105 particles (also known as particle units, pu). Administration of adenovirus or adeno-associated virus.
  • AAV adeno-associated virus
  • the administered amount is at least about 1 ⁇ 10 6 particles, at least about 1 ⁇ 10 7 particles, at least about 1 ⁇ 10 8 particles, or at least about 1 ⁇ 10 9 particles. related virus. Delivery methods and dosages are described, for example, in WO 2016205764 and US Patent No. 8,454,972, which are incorporated herein by reference in their entirety.
  • the vector is a recombinant adeno-associated virus (rAAV) vector.
  • modified AAV vectors can be used for delivery.
  • Modified AAV vectors can be based on one or more of several capsid types, including AAV1, AV2, AAV5, AAV6, AAV8, AAV8.2, AAV9, AAV rh10, modified AAV vectors (e.g., modified AAV2, modified AAV3, modified AAV6) and pseudotyped AAV (such as AAV2/8, AAV2/5 and AAV2/6).
  • Exemplary AAV vectors and techniques that can be used to generate rAAV particles are known in the art (see, e.g., Aponte-Ubillus et al. (2016) Appl. Microbiol. Biotechnol. 102(3): 1045-54; Zhong et al. ( 2012) J. Genet. Syndr. Gene Ther. S1: 008; West et al. (1987) Virology 160: 38-47 (1987); Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-60 ; US Patent Nos. 4,797,368 and 5,173,414; International Publication Nos. WO2015/054653 and WO93/24641, each of which is incorporated herein by reference).
  • AAV vector used to deliver Cas9 and other Cas proteins can be used to deliver the engineered Cas12i system of the present application.
  • the rAAV construct can be administered enterally to a subject. In some embodiments, the rAAV construct can be administered to a subject parenterally. In some embodiments, rAAV particles can be administered subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebroventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, via inhalation, topically, or By direct injection into one or more cells, tissues or organs. In some embodiments, rAAV particles can be administered to a subject by injection into the hepatic artery or portal vein.
  • Vectors can be transferred into host cells by physical, chemical or biological means.
  • vectors into host cells include: calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, eg, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cells by electroporation.
  • Biological methods for introducing heterologous nucleic acids into host cells include the use of DNA and RNA vectors.
  • Viral vectors have become the most widely used method for inserting genes into mammalian, eg human, cells.
  • Chemical methods for introducing vectors into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, Mix micelles and liposomes.
  • An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome (eg, an artificial membrane vesicle).
  • the engineered CRISPR-Cas12i system is delivered in nanoparticles as RNPs.
  • the vector or expression system encoding the CRISPR-Cas12i system or components thereof comprises one or more selectable or detectable markers that provide for the isolation or efficient selection of the containing and/or Means for cells that have been modified by the CRISPR-Cas12i system (e.g. at an early stage and at a large scale).
  • Reporter genes can be used to identify potentially transfected cells and assess the function of regulatory sequences.
  • a reporter gene is a gene that is absent or not expressed in the recipient organism or tissue and whose expression of the encoded polypeptide is evidenced by some readily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at an appropriate time after introduction of the DNA into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g. Ui-Tei et al. FEBS Letters 479: 79-82 (2000)).
  • heterologous nucleic acid in host cells include, for example, molecular biological assays, such as Southern and Northern blots, RT-PCR, and PCR, well known to those skilled in the art; biochemical assays, such as by immunological methods such as ELISA and Western blot) to detect the presence or absence of specific peptides.
  • molecular biological assays such as Southern and Northern blots, RT-PCR, and PCR, well known to those skilled in the art
  • biochemical assays such as by immunological methods such as ELISA and Western blot
  • the nucleic acid sequence encoding the engineered Cas12i effector protein and/or the guide RNA is operably linked to a promoter.
  • the promoter is an endogenous promoter relative to the cell engineered using the engineered CRISPR-Cas12i system.
  • the nucleic acid encoding the engineered Cas12i effector protein can be knocked into the genome of an engineered mammalian cell downstream of an endogenous promoter using any method known in the art.
  • the endogenous promoter is the promoter of an abundant protein such as ⁇ -actin.
  • the endogenous promoter is an inducible promoter, eg, inducible by an endogenous activation signal of the engineered mammalian cell.
  • the promoter is a T cell activation dependent promoter (such as the IL-2 promoter, NFAT promoter or NF ⁇ B promoter).
  • the promoter is a heterologous promoter relative to the cell engineered using the engineered CRISPR-Cas12i system.
  • a variety of promoters have been explored for expression of genes in mammalian cells, and any promoter known in the art may be used in this application. Promoters can be broadly classified as constitutive promoters or regulated promoters, such as inducible promoters.
  • the nucleic acid sequence encoding the engineered Cas12i effector protein and/or the guide RNA is operably linked to a constitutive promoter.
  • Constitutive promoters allow the constitutive expression of heterologous genes (also known as transgenes) in host cells.
  • Exemplary constitutive promoters contemplated herein include, but are not limited to: cytomegalovirus (CMV) promoter, human elongation factor-1 ⁇ (hEF1 ⁇ ), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoter (PGK), The simian virus 40 early promoter (SV40) and the chicken ⁇ -actin promoter are coupled to the CMV early enhancer (CAG).
  • CMV cytomegalovirus
  • hEF1 ⁇ human elongation factor-1 ⁇
  • UbiC ubiquitin C promoter
  • PGK phosphoglycerol kinase promoter
  • the promoter is a CAG promoter comprising a cytomegalovirus (CMV) early enhancer element, a promoter, the first exon and the first intron of the chicken ⁇ -actin gene , and the splice acceptor of the rabbit ⁇ -globin gene.
  • CMV cytomegalovirus
  • the nucleic acid sequence encoding the engineered CRISPR-Cas12i effector protein and/or the guide RNA is operably linked to an inducible promoter.
  • Inducible promoters are a type of regulated promoter.
  • the inducible promoter can be induced by one or more conditions, such as physical conditions, the microenvironment or the physiological state of the host cell, an inducer (ie, an inducer), or a combination thereof.
  • the induction conditions are selected from the group consisting of inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and the CRISPR-Cas12i system to be engineered Activation state of engineered cells.
  • the promoter is inducible by a small molecule inducing agent such as a compound.
  • the small molecule is selected from the group consisting of doxycycline, tetracycline, alcohol, metal, or steroid. Chemically inducible promoters have been most extensively studied.
  • Such promoters include promoters whose transcriptional activity is regulated by the presence or absence of small molecule chemicals such as doxycycline, tetracycline, alcohols, steroids, metals and other compounds.
  • the doxycycline-inducible system with retrotetracycline-controlled transactivator (rtTA) and tetracycline-responsive element promoter (TRE) is currently the most mature system.
  • rtTA retrotetracycline-controlled transactivator
  • TRE tetracycline-responsive element promoter
  • WO9429442 describes the tight control of gene expression in eukaryotic cells by tetracycline-responsive promoters.
  • WO9601313 discloses tetracycline regulated transcriptional regulators.
  • Tet technologies such as the Tet-on system have been described at, for example, the TetSystems.com website.
  • any known chemically regulated promoter can be used to drive the expression of the engineered CRISPR-Cas12i protein
  • the nucleic acid sequence encoding the engineered Cas12i effector protein is codon optimized.
  • an expression construct comprising a codon-optimized sequence encoding the engineered Cas12i effector protein linked to a BPK2104-ccdB vector.
  • the expression construct encodes a tag (eg, 10 ⁇ His tag) operably linked to the C-terminus of the engineered Cas12i effector protein.
  • each engineered split Cas12i construct encodes a fluorescent protein such as GFP or RFP.
  • the reporter protein can be used to assess the co-localization and/or dimerization of the engineered Cas12i protein, for example using microscopy.
  • Nucleic acid sequences encoding engineered Cas12i effector proteins can be fused to nucleic acid sequences encoding additional components using sequences encoding self-cleaving peptides such as T2A, P2A, E2A or F2A peptides.
  • an expression construct for use in mammalian cells comprising a nucleic acid sequence encoding said engineered Cas12i effector protein.
  • the expression construct comprises a codon-optimized sequence encoding the engineered Cas12i effector protein inserted into the pCAG-2A-eGFP vector such that the Cas12i protein is operably linked to eGFP.
  • a second vector is provided for expression of a guide RNA (eg, crRNA or pre-crRNA array) in a mammalian cell (eg, a human cell).
  • the sequence encoding the guide RNA is expressed in the pUC19-U6-i2-cr RNA vector backbone.
  • one or more vectors expressing one or more elements of the CRISPR-Cas12i system are introduced into the host cell such that expression of the elements of the CRISPR-Cas12i system directs the nucleic acid targeting complex at one or more target sites point formation.
  • a Cas12i nucleic acid-targeting effector enzyme and a nucleic acid-targeting guide RNA can each be operably linked to separate regulatory elements on separate vectors.
  • the RNA of the nucleic acid targeting system can be delivered to a transgenic Cas12i nucleic acid targeting effector protein animal or mammal, e.g., an animal or mammal that constitutively or inducibly or conditionally expresses the nucleic acid targeting effector protein; or otherwise expresses the nucleic acid Targeting the effector protein or an animal or mammal having cells containing the nucleic acid targeting effector protein, for example by prior administration thereto of one or more vectors encoding and expressing the nucleic acid targeting effector protein in vivo.
  • two or more elements expressed by the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the nucleic acid targeting system not contained in the first vector.
  • the elements of the nucleic acid targeting system combined in a single vector may be arranged in any suitable orientation, eg, one element is positioned 5' ("upstream") or 3' ("downstream") relative to a second element.
  • the coding sequence for one element may be located on the same or opposite strand and oriented in the same or opposite orientation as the coding sequence for a second element.
  • a single promoter drives the expression of transcripts encoding a Cas12i nucleic acid-targeting effector protein and a nucleic acid-targeting guide RNA that are embedded within one or more intronic sequences (e.g., each in a different Introns, two or more in at least one intron, or all in a single intron).
  • nucleic acid-targeting effector protein and the nucleic acid-targeting guide RNA can be operably linked to and expressed from the same promoter.
  • Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expressing one or more elements of the nucleic acid targeting system are as used in aforementioned documents such as WO 2014/093622 (PCT/US2013/074667).
  • a vector comprises one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites").
  • one or more insertion sites are located at one Upstream and/or downstream of one or more sequence elements of one or more vectors.
  • a single expression construct can be used to target nucleic acid targeting activity to multiple different corresponding target sequences within a cell.
  • a single vector may comprise about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more guide sequences.
  • about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more such guide sequence-containing vectors may be provided and optionally delivered to cell.
  • the vector comprises a regulatory element operably linked to an enzyme coding sequence encoding a nucleic acid targeting effector protein.
  • the Cas12i nucleic acid-targeting effector protein or one or more nucleic acid-targeting guide RNAs may be delivered separately; and advantageously, at least one of these is delivered via a particle complex.
  • the nucleic acid-targeting effector mRNA can be delivered before the nucleic acid-targeting guide RNA to allow time for expression of the Cas12i nucleic acid-targeting effector.
  • the nucleic acid-targeting effector protein mRNA can be administered 1-12 hours (preferably about 2-6 hours) prior to administration of the nucleic acid-targeting guide RNA.
  • nucleic acid-targeting effector protein mRNA and nucleic acid-targeting guide RNA can be administered together.
  • the second booster dose of guide RNA may be administered 1-12 hours (preferably about 2-6 hours) after the initial administration of nucleic acid-targeting effector protein mRNA+guide RNA. Additional administration of nucleic acid-targeting effector protein mRNA and/or guide RNA may be useful to achieve the most efficient level of genome modification.
  • a CRISPR-Cas12i system comprising: (1) any of the engineered Cas12i effector proteins (such as any of the engineered Cas12i nucleases or functional variants thereof) or a polynucleotide encoding any of the engineered Cas12i effector proteins; and (2) a crRNA or a polynucleotide encoding the crRNA comprising: (i) a spacer capable of hybridizing to a target sequence of a target DNA sequence, and (ii) a direct repeat sequence linked to the spacer sequence capable of directing the engineered Cas12i effector protein to bind to the crRNA to form a CRISPR-Cas12i complex targeting the target sequence.
  • a CRISPR-Cas12i system comprising one or more vectors, said one or more vectors comprising: (1) a first regulatory element, said first regulatory element being operably linked To the nucleotide sequence of any one of the engineered Cas12i effector proteins (such as any one of the engineered Cas12i nucleases or functional variants thereof); and (2) a second regulatory element, the second regulatory element
  • the element is operably linked to a polynucleotide encoding a crRNA comprising: (i) a spacer sequence capable of hybridizing to a target sequence of a target DNA, and (ii) a spacer sequence linked to the spacer sequence capable of directing the engineered
  • the Cas12i effector protein binds to the crRNA to form a direct repeat of the CRISPR-Cas12i complex targeting the target sequence; wherein the first regulatory element and the second regulatory element are located at the CRISPR-Cas12i system on the same or different carriers.
  • the first regulatory element and the second regulatory element are located on different vectors of the CRISPR-Cas12i system. In certain embodiments, the first regulatory element and the second regulatory element are located on the same vector of the CRISPR-Cas12i system. In certain embodiments, the first regulatory element and the nucleotide sequence encoding the engineered Cas12i effector protein are upstream of the second regulatory element and the polynucleotide encoding crRNA. In certain embodiments, the first regulatory element and the nucleotide sequence encoding the engineered Cas12i effector protein are downstream of the second regulatory element and the polynucleotide encoding crRNA. In certain embodiments, the first regulatory element and the second regulatory element are the same. In certain embodiments, said first regulatory element and said second regulatory element are different.
  • a CRISPR-Cas12i system which includes a carrier, and the carrier comprises: (1) encoding any of the engineered Cas12i effector protein (such as any of the engineered Cas12i nucleic acid (2) a polynucleotide encoding a crRNA comprising: (i) a spacer sequence capable of hybridizing to a target sequence of a target DNA, and (ii) linked to the A direct repeat of a spacer sequence capable of directing the engineered Cas12i effector protein to bind to the crRNA to form a CRISPR-Cas12i complex targeting the target sequence; and (3) a regulatory element operable is connected to the polynucleotide encoding the engineered Cas12i effector protein and the polynucleotide encoding the crRNA.
  • the vector comprises the regulatory element, the polynucleotide encoding the engineered Cas12i effector protein and the polynucleotide encoding the crRNA from 5' to 3'. In certain embodiments, the vector comprises the regulatory element, the polynucleotide encoding the crRNA and the polynucleotide encoding the engineered Cas12i effector protein from 5' to 3'.
  • the polynucleotide encoding the crRNA and the polynucleotide encoding the engineered Cas12i effector protein are connected by a linker sequence, such as encoding P2A, T2A, E2A, F2A, BmCPV 2A, BmIFV 2A, (GS)n (SEQ ID NO:74), (GGGS)n (SEQ ID NO:75) and (GGGGS)n (SEQ ID NO:76) in any polynucleotide sequence (wherein n is at least 1), or any polynucleotide sequence of IRES, SV40, CMV, UBC, EF1 ⁇ , PGK and CAGG, or any combination thereof.
  • a linker sequence such as encoding P2A, T2A, E2A, F2A, BmCPV 2A, BmIFV 2A, (GS)n (SEQ ID NO:74), (GGGS)n (SEQ ID NO:75) and (GGGGS)
  • the components of the CRISPR-Cas12i system of the present invention can be delivered in various forms, such as a combination of DNA/RNA or RNA/RNA or protein RNA.
  • an engineered Cas12i effector protein such as any one of the engineered Cas12i nucleases or a functional variant thereof can be delivered as a DNA-encoding polynucleotide or an RNA-encoding polynucleotide or as a protein.
  • the guides can be delivered as DNA-encoding polynucleotides or as RNA. Mixed delivery forms can be used.
  • the invention provides a method comprising delivering one or more polynucleotides, e.g., one or more vectors as described herein, one or more transcripts thereof, and/or one or more proteins transcribed therefrom, to Host Cell Methods.
  • one or more polynucleotides e.g., one or more vectors as described herein, one or more transcripts thereof, and/or one or more proteins transcribed therefrom, to Host Cell Methods.
  • the application provides the use of any of the engineered Cas12i effector proteins described herein (such as any of the engineered Cas12i nucleases or functional variants thereof) or the CRISPR-Cas12i system to detect targets in vitro, ex vivo or in vivo Nucleic acids or methods of modifying nucleic acids, and methods of using said engineered Cas12i effector protein or CRISPR-Cas12i system for therapy (such as gene editing) or diagnosis.
  • any of the engineered Cas12i effector proteins described herein such as any of the engineered Cas12i nucleases or functional variants thereof
  • CRISPR-Cas12i system to detect targets in vitro, ex vivo or in vivo Nucleic acids or methods of modifying nucleic acids, and methods of using said engineered Cas12i effector protein or CRISPR-Cas12i system for therapy (such as gene editing) or diagnosis.
  • engineered Cas12i effector proteins or CRISPR-Cas12i systems described herein for detecting or modifying nucleic acids in cells, and for treating or diagnosing a disease or condition in a subject; and comprising said engineered Compositions of any one of the Cas12i effector proteins or one or more components of the engineered CRISPR-Cas12i system are useful in the preparation of nucleic acids for detection or modification of cells and for the treatment or diagnosis of diseases in subjects Use in medicine for a medical condition or condition.
  • the present application also provides methods for detecting target nucleic acids using any of the engineered Cas12i effector proteins or CRISPR-Cas12i systems with improved activity.
  • the use of Cas12i effector proteins as detection reagents can take advantage of the discovery that, once activated by the detection of target DNA, type V CRISPR/Cas proteins (e.g., Cas12i) can promiscuously cleave non-targeted single-stranded DNA (ssDNA or RNA, i.e. guide A single-stranded nucleic acid to which the guide sequence of RNA does not hybridize).
  • target DNA double-stranded or single-stranded
  • a sample e.g., in some cases above a threshold amount
  • the result is cleavage of the single-stranded nucleic acid in the sample, which can be detected using any convenient detection method (for example, using tagged single-stranded detection nucleic acids such as DNA or RNA).
  • Cas12i can cut ssDNA and ssRNA.
  • methods using Cas proteins as detection reagents are described in US10253365 and WO2020/056924, which are hereby incorporated by reference in their entirety.
  • a method of detecting target DNA comprising: (a) contacting the sample with: (i) an engineered DNA described herein Cas12i effector protein (such as any one of said engineered Cas12i nuclease or its functional variant); (ii) guide RNA, which comprises a guide sequence hybridized with said target DNA; and (iii) detection nucleic acid , which is single-stranded (i.e., "single-stranded detection nucleic acid”) and does not hybridize to the guide sequence of the guide RNA; and (b) measuring the generation of the single-stranded detection nucleic acid by the engineered Cas12i effector protein detectable signal.
  • an engineered DNA described herein Cas12i effector protein such as any one of said engineered Cas12i nuclease or its functional variant
  • guide RNA which comprises a guide sequence hybridized with said target DNA
  • detection nucleic acid which is single-stranded (i.e., "single-stranded detection nu
  • the single-stranded detection nucleic acid includes a fluorescence emitting dye pair (eg, the fluorescence emitting dye pair is a fluorescence resonance energy transfer (FRET) pair, a quencher/fluorescent pair).
  • the target DNA is viral DNA (eg, papillomavirus, hepadnavirus, herpesvirus, adenovirus, poxvirus, parvovirus, etc.).
  • the single-stranded detection nucleic acid is DNA.
  • the single-stranded detection nucleic acid is RNA.
  • the method for detecting target DNA (single-stranded or double-stranded) in a sample of the present disclosure can detect target DNA with high sensitivity.
  • the methods of the present disclosure can be used to detect target DNA present in a sample comprising a plurality of DNAs, including the target DNA and a plurality of non-target DNAs, wherein the target DNA is expressed in the order of 107 non-target DNA
  • One or more copies of target DNA present eg, one or more copies per 10 6 non-target DNA, one or more copies per 10 5 non-target DNA, one or more copies per 10 4
  • the engineered Cas12i effector proteins described herein can detect target DNA with greater sensitivity than the reference Cas12i nuclease. In some embodiments, compared to the reference Cas12i nuclease, the engineered Cas12i effector protein can be 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% , 70%, 80%, 90%, 95% or higher sensitivity to detect target DNA.
  • the present application provides methods of modifying a target nucleic acid comprising a target sequence comprising contacting the target nucleic acid with any of the engineered CRISPR-Cas12i systems described herein.
  • the methods are performed in vitro.
  • the target nucleic acid is present in a cell.
  • the cells are bacterial cells, yeast cells, mammalian cells, plant cells, or animal cells.
  • the methods are performed ex vivo.
  • the methods are performed in vivo.
  • Target nucleic acid modification includes, but is not limited to, target nucleic acid single-strand cleavage, double-strand cleavage, base substitution, base insertion, base deletion, mutation (such as pathogenic mutation) sequence repair, etc.
  • the target nucleic acid is cleaved or a target sequence in the target nucleic acid is altered by the engineered CRISPR-Cas12i system. In some embodiments, expression of the target nucleic acid is altered by the engineered CRISPR-Cas12i system. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target sequence is associated with a disease or condition, eg, based on misexpression (eg, overexpression, nonexpression, or expression of a pathogenic RNA or protein) of the target sequence. In some embodiments, the engineered CRISPR-Cas12i system comprises an array of precursor guide RNAs encoding multiple crRNAs, wherein each crRNA comprises a different guide sequence.
  • the present application provides a method of treating a disease or condition associated with a target nucleic acid in a cell of an individual comprising modifying the target nucleic acid in the cell of the individual using any of the methods described herein, The disease or condition is thereby treated.
  • the disease or condition is selected from the group consisting of cancer, cardiovascular disease, genetic disease (e.g., a gene defect disease such as sickle cell anemia (SCD) or beta-thalassemia (TDT)), Autoimmune diseases, metabolic diseases, neurodegenerative diseases, eye diseases, bacterial and viral infections.
  • SCD sickle cell anemia
  • TTT beta-thalassemia
  • the engineered CRISPR-Cas12i system described herein can modify target nucleic acids in cells in a variety of ways, depending on the type of Cas12i effector protein engineered in the CRISPR-Cas12i system.
  • the methods induce site-specific cleavage in the target nucleic acid.
  • the methods cleave genomic DNA in cells such as bacterial cells, plant cells, or animal cells (eg, mammalian cells).
  • the method kills the cell by cleaving the genomic DNA in the cell.
  • the methods cleave viral nucleic acid in the cell.
  • the methods alter (eg, increase or decrease) the expression level of the target nucleic acid in a cell.
  • the method uses an engineered Cas12i effector protein to increase the expression level of the target nucleic acid in a cell, eg, based on an enzymatically inactive Cas12i protein fused to a transactivation domain.
  • the method uses an engineered Cas12i effector protein to reduce the expression level of the target nucleic acid in a cell, eg, based on an enzymatically inactive Cas12i protein fused to a transcriptional repression domain.
  • the method is based, for example, on an enzymatically inactive Cas12i protein fused to an epigenetic modification domain, using an engineered Cas12i effector protein to introduce epigenetic modifications into the target nucleic acid or target nucleic acid in the cell Associated proteins (eg, proteins that bind to the target nucleic acid or are in its vicinity, such as transcription factors or histones).
  • an engineered Cas12i effector protein to introduce epigenetic modifications into the target nucleic acid or target nucleic acid in the cell Associated proteins (eg, proteins that bind to the target nucleic acid or are in its vicinity, such as transcription factors or histones).
  • the engineered Cas12i system described herein can be used to introduce other modifications into the target nucleic acid, depending on the functional domains comprised by the engineered Cas12i effector protein.
  • the methods alter a target sequence in the target nucleic acid in a cell.
  • the method introduces a mutation into the target nucleic acid in the cell, such as mutating a pathogenic mutation into a non-pathogenic sequence (e.g., using a Any dCas12i) described in the present invention.
  • the methods use one or more endogenous DNA repair pathways, such as non-homologous end joining (NHEJ) or homology-directed recombination (HDR), to repair the double DNA induced in the target DNA in the cell. Strand breaks, as a result of sequence-specific cleavage by the CRISPR complex.
  • NHEJ non-homologous end joining
  • HDR homology-directed recombination
  • Exemplary mutations include, but are not limited to, insertions, deletions, substitutions, and frameshifts.
  • pegRNA is used to simultaneously perform target cleavage and provide a template for DNA repair.
  • the methods insert donor DNA at the target site.
  • insertion of donor DNA results in the introduction of a selectable marker or reporter protein into the cell.
  • insertion of donor DNA results in knock-in of the gene.
  • insertion of donor DNA results in a knockout mutation.
  • insertion of donor DNA results in substitutional mutations such as single nucleotide substitutions.
  • the method induces a phenotypic change in the cell.
  • the engineered CRISPR-Cas12i system is used as part of a genetic circuit, or to insert a genetic circuit into the genomic DNA of a cell.
  • the inducer-controlled engineered split-type Cas12i effector proteins described herein are particularly useful as components of genetic circuits.
  • Genetic circuits can be used in gene therapy. Methods and techniques for designing and using genetic circuits are known in the art. Further reference can be made to eg Brophy, Jennifer AN, and Christopher A. Voigt. "Principles of genetic circuit design.” Nature methods 11.5 (2014): 508.
  • the target nucleic acid is in a cell.
  • the target nucleic acid is genomic DNA.
  • the target nucleic acid is extrachromosomal DNA.
  • the target nucleic acid is foreign to the cell.
  • the target nucleic acid is viral nucleic acid such as viral DNA.
  • the target nucleic acid is a plasmid in a cell.
  • the target nucleic acid is a horizontally transferred plasmid.
  • the target nucleic acid is RNA.
  • the target nucleic acid is an isolated nucleic acid such as isolated DNA. In some embodiments, the target nucleic acid is present in a cell-free environment. In some embodiments, the target nucleic acid is an isolated vector such as a plasmid. In some embodiments, the target nucleic acid is an isolated linear DNA fragment.
  • the cells are bacteria, yeast cells, fungal cells, algal cells, plant cells, or animal cells (eg, mammalian cells, such as human cells).
  • the cells are of natural origin such as cells isolated from a tissue biopsy.
  • the cells are cells isolated from cell lines cultured in vitro.
  • the cells are from a primary cell line.
  • the cells are from an immortalized cell line.
  • the cells are genetically engineered cells.
  • the cell is an animal cell of an organism selected from the group consisting of cattle, sheep, goats, horses, pigs, deer, chickens, ducks, geese, rabbits, and fish.
  • the cell is a plant cell of an organism selected from the group consisting of corn, wheat, barley, oat, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, pearl millet , corn, sorghum, canola, hemp, vegetable crops, forage crops, industrial crops, tree crops and biomass crops.
  • the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the human cells are human embryonic kidney 293T (HEK293T or 293T) cells or HeLa cells. In some embodiments, the cells are human embryonic kidney (HEK293T) cells. In some embodiments, the cells are mouse Hepal-6 cells. In some embodiments, the mammalian cells are selected from the group consisting of immune cells, liver cells, tumor cells, stem cells, blood cells, nerve cells, zygotes, muscle cells (eg, cardiomyocytes), and skin cells.
  • HEK293T human embryonic kidney 293T
  • the cells are mouse Hepal-6 cells.
  • the mammalian cells are selected from the group consisting of immune cells, liver cells, tumor cells, stem cells, blood cells, nerve cells, zygotes, muscle cells (eg, cardiomyocytes), and skin cells.
  • the cells are immune cells selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) T cells, iNK-T cells, NK-T-like cells, ⁇ T cells, tumor Infiltrating T cells and dendritic cell (DC) activated T cells.
  • the methods generate modified immune cells, such as CAR-T cells or TCR-T cells.
  • the cells are embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, progenitor cells of gametes, cells in gametes, zygotes, or embryos.
  • ES embryonic stem
  • iPS induced pluripotent stem
  • the methods described herein can be used to modify target cells in vivo, ex vivo, or in vitro, and can be done in such a way that the cells are altered such that, once modified, progeny or cell lines of the modified cells retain the modified Altered phenotype.
  • the modified cells and progeny may be part of a multicellular organism such as a plant or animal with ex vivo or in vivo applications such as genome editing and gene therapy.
  • the methods are performed ex vivo.
  • the modified cells eg, mammalian cells
  • the modified cells are propagated ex vivo.
  • the modified cells are cultured to propagate for at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days .
  • the modified cells are cultured for no more than about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days.
  • the modified cells are further evaluated or screened to select cells with one or more desired phenotypes or properties.
  • the target sequence is a sequence associated with a disease or condition.
  • diseases or conditions include, but are not limited to, cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection.
  • the disease or condition is a genetic disease.
  • the disease or condition is a monogenic disease or condition.
  • the disease or condition is a polygenic disease or condition.
  • the target sequence has a mutation compared to the wild-type sequence. In some embodiments, the target sequence has a single nucleotide polymorphism (SNP) associated with a disease or condition.
  • SNP single nucleotide polymorphism
  • the donor DNA inserted into the target nucleic acid encodes a biological product selected from the group consisting of reporter proteins, antigen-specific receptors, therapeutic proteins, antibiotic resistance proteins, RNAi molecules, cytokines, kinases , antigens, antigen-specific receptors, cytokine receptors, and suicide polypeptides.
  • the donor DNA encodes a therapeutic protein.
  • the donor DNA encodes a therapeutic protein useful in gene therapy.
  • the donor DNA encodes a therapeutic antibody.
  • the donor DNA encodes an engineered receptor, such as a chimeric antigen receptor (CAR) or an engineered TCR.
  • the donor DNA encodes a therapeutic RNA, such as a small RNA (eg, siRNA, shRNA, or miRNA) or a long non-coding RNA (lincRNA).
  • the methods described herein can be used for multiplex gene editing or modulation at two or more (e.g., 2, 3, 4, 5, 6, 8, 10 or more) different target sites.
  • the methods detect or modify multiple target nucleic acids or target nucleic acid sequences.
  • the method comprises contacting the target nucleic acid with a guide RNA comprising a plurality (e.g., 2, 3, 4, 5, 6, 8, 10, or more) of crRNA sequences, wherein each crRNA comprises different target sequences.
  • engineered cells comprising a modified target nucleic acid produced using any of the methods described herein.
  • the engineered cells can be used in cell therapy.
  • Autologous or allogeneic cells can be used to prepare engineered cells using the methods described herein for cell therapy.
  • the methods described herein can also be used to generate isogenic lines of cells (eg, mammalian cells) to study genetic variants.
  • engineered non-human animals comprising engineered cells described herein.
  • the engineered non-human animal is a genome-edited non-human animal.
  • the engineered non-human animals can be used as disease models.
  • Non-human genome edited or transgenic animals include, but are not limited to: pronuclear microinjection, viral infection, transformation of embryonic stem cells and induced pluripotent stem (iPS) cells.
  • iPS induced pluripotent stem
  • Detailed methods that can be used include, but are not limited to, those described by Sundberg and Ichiki (2006, Genetically Engineered Mice Handbook, CRC Press) and those described by Gibson (2004, A Primer Of Genome Science 2nd ed. Sunderland, Mass.: Sinauer).
  • the engineered animal can be of any suitable species including, but not limited to: cattle, horses, sheep, dogs, deer, felines, goats, pigs, primates, and lesser known mammals such as elephants , deer, zebra or camel.
  • a use of the aforementioned engineered CRISPR-Cas12i system in preparing a medicament for treating a disease or disorder associated with a target nucleic acid in cells of an individual is provided.
  • methods of the aforementioned engineered CRISPR-Cas12i system in treating a disease or disorder associated with a target nucleic acid in cells of an individual are provided.
  • the present invention provides a method of treating a disease in a subject (such as a human) in need thereof, the method comprising administering (such as intravenous injection or infusion) to the subject a drug of the present invention.
  • CRISPR-Cas12i system it comprises: (1) Cas12i effector protein of any described engineering (such as Cas12i nuclease or functional variant thereof of arbitrary described engineering), or the Cas12i effector protein of encoding described engineering and (2) crRNA, or a polynucleotide encoding said crRNA, said crRNA comprising: (i) a spacer sequence capable of hybridizing to a target sequence on a target nucleic acid associated with the disease, and (ii ) a direct repeat sequence linked to the spacer sequence capable of guiding the engineered Cas12i effector protein to bind to the crRNA to form a CRISPR-Cas12i complex targeting the target sequence; wherein the spacer sequence and the Hybridization of the target sequence media
  • the present application provides methods of treating a disease or condition associated with a target nucleic acid in a cell of an individual comprising contacting the target nucleic acid with any of the engineered CRISPR-Cas12i systems described herein, wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid, wherein the engineered Cas12i effector protein and the guide RNA associate with each other to bind to the target nucleic acid to modify (such as cutting or base replacing) said target nucleic acid, thereby allowing said disease or condition to be treated.
  • mutations are introduced into the target nucleic acid.
  • expression of the target nucleic acid is enhanced.
  • expression of the target nucleic acid is inhibited.
  • the present application provides methods of treating a disease or condition in an individual comprising administering to the individual an effective amount of any of the engineered CRISPR-Cas12i systems described herein and a donor encoding a therapeutic agent DNA (such as a non-pathogenic native sequence used as a repair template), wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid of the individual, wherein the engineered Cas12i effector protein and the guide RNA are mutually Binding to bind to the target nucleic acid and insert donor DNA into the target sequence, thereby allowing the disease or condition to be treated.
  • a therapeutic agent DNA such as a non-pathogenic native sequence used as a repair template
  • the present application provides a method of treating a disease or condition in an individual comprising administering to the individual an effective amount of an engineered cell comprising a modified target nucleic acid, wherein the engineered cell is obtained by Prepared by contacting the cells with any of the engineered CRISPR-Cas12i systems described herein, wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid, wherein the engineered Cas12i effects The protein and the guide RNA associate with each other to bind to the target nucleic acid to modify the target nucleic acid.
  • the engineered cells are immune cells.
  • the engineered cells are stem cells (eg, hematopoietic stem cells, neural stem cells).
  • the engineered cells are neural cells.
  • the individual is a human.
  • the individual is an animal, eg, a model animal such as a rodent, pet, or farm animal.
  • the individual is a mammal, such as a cat, dog, rabbit, hamster, and the like.
  • the disease or condition is selected from the group consisting of cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection.
  • the target nucleic acid is PCSK9.
  • the disease or condition is cardiovascular disease.
  • the disease or condition is coronary artery disease.
  • the method reduces cholesterol levels in the individual.
  • the method treats diabetes in the individual.
  • CRISPR-Cas12i system of the present invention can be used to treat include, but are not limited to, cystic fibrosis, hereditary angioedema, diabetes, progressive pseudohypertrophic muscular dystrophy, Becker muscular dystrophy, alpha-1- Antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, high Lipidemia, hypercholesterolemia, Leber's congenital amaurosis, sickle cell disease, or beta thalassemia, etc.
  • the condition or disease is transthyretin amyloidosis, such as wild-type transthyretin amyloidosis (transthyretin-related wild-type amyloidosis, ATTRwt), hereditary transthyretin amyloidosis transthyretin-related hereditary amyloidosis (ATTRm), familial amyloid polyneuropathy (FAP, ATTR-PN), or familial amyloid cardiomyopathy (FAC, ATTR-CM) .
  • the disorder or disease is transthyretin instability caused by TTR gene mutation or abnormal expression (such as high expression).
  • the disorder or disease is other disorder or disease caused by TTR gene mutation or abnormal expression (such as high expression), or a derivative disorder or disease.
  • the engineered CRISPR-Cas12i system described herein, or components thereof, nucleic acid molecules thereof, or nucleic acid molecules encoding or providing components thereof can be delivered to host cells by various delivery systems such as plasmids or viruses (for example, any of the vectors described in the "Constructs and Vectors" section above).
  • the engineered CRISPR-Cas12i system can be delivered by other means, such as a ribonucleoprotein complex consisting of the engineered Cas12i effector protein and its one or more cognate RNA guide sequences nucleofection or electroporation.
  • delivery is via nanoparticles or exosomes.
  • paired Cas12i nickase complexes can be delivered directly using nanoparticles or other direct protein delivery methods such that complexes comprising two paired crRNA elements are co-delivered.
  • proteins can be delivered to cells via viral vectors or directly followed by CRISPR arrays containing two paired spacers for double nicks.
  • the RNA can be conjugated to at least one sugar moiety such as N-acetylgalactosamine (GalNAc) (particularly a three-antennary GalNAc).
  • the engineered CRISPR-Cas12i system can be delivered using any delivery method suitable for the disease being treated, such as delivery by intravenous injection or drip, or local delivery at the diseased site (such as tumor delivered within).
  • Suitable routes of administration of the engineered CRISPR-Cas12i systems described herein include, but are not limited to: topical, subcutaneous, transdermal, intradermal, intralesional, intra-articular, intraperitoneal, intravesical, transmucosal, gingival, intradental, cochlear Intratympanic, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral and intraventricular administration.
  • the engineered CRISPR-Cas12i system described herein is administered to a subject by injection, by catheter, by suppository, or by implant, which is a porous, non-porous, or gel-like material comprising Membranes such as sialic acid membranes, or fibers.
  • compositions comprising one or more components of any of the engineered Cas12i nucleases described herein or functional variants thereof, engineered Cas12i effector proteins or engineered CRISPR-Cas12i systems are also provided Boxes, unit doses and articles of manufacture.
  • kits comprising: one or more AAV vectors encoding the engineered Cas12i nucleases described herein or functional variants thereof, engineered Cas12i effector proteins, or engineered CRISPR-Cas12i systems any of these.
  • the kit further comprises one or more guide RNAs (or DNAs or vectors encoding them).
  • the kit further comprises donor DNA.
  • the kit further comprises cells such as human cells.
  • the kit may comprise one or more additional components, such as containers, reagents, media, cytokines, buffers, antibodies, etc., to allow propagation of the engineered cells.
  • the kit may also comprise a device for administering the composition.
  • the kit may also comprise instructions for using the engineered CRISPR-Cas12i system described herein, such as methods for detecting or modifying target nucleic acids.
  • the kit comprises instructions for treating or diagnosing the disease or condition.
  • the instructions pertaining to the use of the kit components will generally include information on the amount to be administered, the schedule of administration and the route of administration for such deliberate treatment.
  • the container can be a unit dose, bulk package (eg, a multi-dose package), or a subunit dose.
  • kits comprising sufficient doses of a composition disclosed herein can be provided to provide effective treatment of an individual over an extended period of time.
  • the kit can also include a plurality of unit doses of the composition and instructions for use packaged in quantities sufficient for storage and use in a pharmacy (eg, hospital pharmacy and compounding pharmacy).
  • kits of the invention are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and explanatory information. Accordingly, the present application also provides an article of manufacture including vials (eg, sealed vials), bottles, jars, flexible packaging, and the like.
  • the article of manufacture may comprise a container and a label or package insert on or adhered to the container.
  • Suitable containers include, for example, bottles, vials, syringes, and the like.
  • the container can be formed from a variety of materials such as glass or plastic.
  • the container contains a composition effective to treat a disease or condition described herein, and may have a sterile access port (e.g., the container may be a bag of intravenous solution or a vial with a stopper pierceable by a hypodermic needle) .
  • the label or package insert indicates that the composition is used to treat a particular condition in an individual.
  • the label or package insert will further include instructions for administering the composition to the individual.
  • Package Insert means the instructions commonly included in commercial packages of therapeutic products that contain information on the indications, usage, dosage, administration, contraindications and/or warnings regarding the use of such therapeutic products.
  • the article of manufacture may also include a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution.
  • a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate buffered saline such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution.
  • BWFI bacteriostatic water for injection
  • the ingredients are presented individually or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet showing the active dose.
  • a hermetically sealed container such as an ampoule or sachet showing the active dose.
  • the drug When the drug is administered by infusion, it can be dispensed with an infusion bottle filled with sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • Example 1 Replace the amino acid that interacts with PAM in the reference Cas12i2 enzyme with a positively charged amino acid, and verify its gene editing efficiency.
  • the coding sequence of Cas12i2 was codon optimized (human) and synthesized. Variants of the Cas protein were generated by PCR-based site-directed mutagenesis. The specific method is to divide the DNA sequence design of the Cas12i2 protein into two parts centered on the mutation site, design two pairs of primers to amplify the two parts of the DNA sequence, and introduce the sequence to be mutated into the primers, and finally clone by Gibson Both fragments were loaded onto the pCAG-2A-eGFP vector (with penicillin resistance). The combination of mutants is constructed by splitting the DNA of the Cas12i2 protein into multiple segments and using PCR and Gibson clone.
  • the position of the mutant is determined by analyzing the structural information of Cas12i2 using protein structure visualization software commonly used in the art (for example, PyMol, Chimera, etc. software can be selected).
  • the structural information of Cas12i2 refers to PDB: 6LTU, 6LTR, 6LU0, 6LTP).
  • Cas12i2 effector protein was expressed in human 293T cells by pCAG-2A-eGFP vector.
  • the DNA encoding the Cas12i2 protein was inserted between XmaI and NheI.
  • a vector expressing the crRNA of Cas12i2 in 293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-i2-crRNA backbone.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM Gibco
  • Gibco penicillin-streptomycin
  • Gibco fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • 600ng of a plasmid encoding Cas12i2 protein and 300ng of a plasmid encoding crRNA were transfected into each 24-well-cell culture dish.
  • HEK293T cells to be subjected to fluorescence-activated cell sorting were digested with trypsin-EDTA (0.05%) (Gibco).
  • Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP channel.
  • FACS-sorted GFP-positive HEK293T cells were lysed with buffer L and incubated at 55 °C for 3 h, then at 95 °C for 10 min.
  • dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers.
  • target loci are directly amplified by barcoded PCR using cell lysates as templates.
  • PCR products were purified and pooled into several libraries for high-throughput sequencing.
  • the frequency (%) of indels was analyzed using CRISPResso2 software by calculating the ratio of reads containing indels or indels. In this application, the index of indel frequency (%) is uniformly used to compare and analyze gene editing efficiency. Reads with an amount less than 0.05% of complete reads were discarded.
  • Example 1-A selected four engineered Cas12i2 with a single amino acid substitution
  • the engineered Cas12i2 enzymes with a single mutation in the amino acid sequence were respectively expressed, and the preferred amino acid replacement method and its corresponding gene editing efficiency are shown in Figure 1a, Figure 1b and Table 1.
  • Example 1-B compares the engineered Cas12i2 with multiple preferred amino acid substitutions
  • Table 1 and Figure 1b We combined the point mutations in the four efficiency-enhancing mutants, E176R, K238R, T447R and E563R, which were screened in Example 1-A.
  • Figure 1b and Table 1 by comparing the gene editing efficiency of these mutants and wild-type Cas12i2 at 3 genomic loci: CCR5-3, CCR5-5, and RNF2-7 in 293T cells, we found point mutations After the combination, mutants with further improved efficiency can be obtained.
  • the coding sequences of the crRNA spacers for CCR5-3, RNF2-7 and CCR5-5 used here are shown in SEQ ID NO: 60, 61 and 62, respectively.
  • Example 2 Replace the amino acids involved in opening the double-strand of DNA in the reference Cas12i2 enzyme with amino acids with aromatic rings, and verify their gene editing efficiencies
  • the variant of Cas12i2 protein was produced by site-directed mutagenesis based on PCR.
  • the specific method was to divide the DNA sequence design of Cas12i2 protein into two parts centered on the mutation site, and design two pairs of primers to amplify the two parts of DNA sequence respectively.
  • the sequence to be mutated was introduced into the primer, and finally the two fragments were loaded into the pCAG-2A-eGFP vector by means of Gibson clone.
  • the determination of the amino acid substitution position can be obtained by analyzing the structural information of Cas12i2 using common protein structure visualization software (for example, software such as PyMol and Chimera can be used).
  • Cas12i2 refers to PDB: 6LTU, 6LTR, 6LU0, 6LTP).
  • Cas12i2 effector protein was expressed in human 293T cells by pCAG-2A-eGFP vector.
  • the DNA encoding the Cas12i2 protein was inserted between XmaI and NheI.
  • a vector expressing Cas12i2crRNA in 293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-i2-crRNA backbone.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well-cell culture dishes (Corning) for 16 hours until the cell density reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of a plasmid encoding Cas12i2 protein and 300ng of a plasmid encoding crRNA were transfected into each 24-well-cell culture dish. After transfection68, HEK293T cells to be fluorescence-activated cell sorting (FACS) were digested with trypsin-EDTA (0.05%) (Gibco). Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP channel.
  • FACS fluorescence-activated cell sorting
  • FACS-sorted GFP-positive HEK293T cells were lysed with buffer L and incubated at 55 °C for 3 h, then at 95 °C for 10 min.
  • dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers.
  • target loci are directly amplified by barcoded PCR using cell lysates as templates.
  • PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of indels was analyzed using CRISPResso2 software by calculating the ratio of reads containing indels or indels. Reads with an amount less than 0.05% of complete reads were discarded.
  • Example 3 Replace the amino acid located in the RuvC domain and interacting with the single-stranded DNA substrate in the reference Cas12i2 enzyme with a positively charged amino acid, and verify its gene editing efficiency
  • the variant of Cas12i2 protein was produced by site-directed mutagenesis based on PCR.
  • the specific method was to divide the DNA sequence design of Cas12i2 protein into two parts centered on the mutation site, and design two pairs of primers to amplify the two parts of DNA sequence respectively.
  • the sequence to be mutated was introduced into the primer, and finally the two fragments were loaded into the pCAG-2A-eGFP vector by means of Gibson clone.
  • the combination of mutants is constructed by splitting the DNA of the Cas12i2 protein into multiple segments and using PCR and Gibson clone.
  • the location of the mutant is determined by analyzing the structural information of Cas12i2 with commonly used protein structure visualization software (for example, PyMol, Chimera and other software can be used).
  • the structural information of Cas12i2 refers to PDB ID: 6LTU, 6LTR, 6LU0, 6LTP.
  • the ssDNA substrate shown in these Cas12i2 structures is only 5nt.
  • Cas12i2 effector protein was expressed in human 293T cells by pCAG-2A-eGFP vector.
  • the DNA encoding the Cas12i2 protein was inserted between XmaI and NheI.
  • a vector expressing Cas12i2crRNA in 293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-i2-crRNA backbone.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • the coding sequences of the crRNA spacers for CCR5-3 and RNF2-7 are shown in SEQ ID NO: 60 and 61, respectively.
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM Gibco
  • Gibco penicillin-streptomycin
  • Gibco fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • 600ng of a plasmid encoding Cas12i2 protein and 300ng of a plasmid encoding crRNA were transfected into each 24-well-cell culture dish.
  • HEK293T cells to be subjected to fluorescence-activated cell sorting were digested with trypsin-EDTA (0.05%) (Gibco).
  • Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP channel.
  • FACS-sorted GFP-positive HEK293T cells were lysed with buffer L and incubated at 55 °C for 3 h, then at 95 °C for 10 min.
  • dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers.
  • target loci are directly amplified by barcoded PCR using cell lysates as templates.
  • PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of indels was analyzed using CRISPResso2 software by calculating the ratio of reads containing indels or indels. Reads with an amount less than 0.05% of complete reads were discarded.
  • *L439(L+GG) means that two glycines are inserted after the 439th amino acid.
  • Example 4 Replace the amino acid in the reference Cas12i2 enzyme that interacts with the DNA-RNA double helix with a positively charged amino acid, and verify its gene editing efficiency
  • the variant of Cas12i2 protein was produced by site-directed mutagenesis based on PCR.
  • the specific method was to divide the DNA sequence design of Cas12i2 protein into two parts centered on the mutation site, and design two pairs of primers to amplify the two parts of DNA sequence respectively.
  • the sequence to be mutated was introduced into the primer, and finally the two fragments were loaded into the pCAG-2A-eGFP vector by means of Gibson clone.
  • the combination of mutants is constructed by splitting the DNA of the Cas12i2 protein into multiple segments and using PCR and Gibson clone.
  • the location of the mutant is determined by analyzing the structural information of Cas12i2 with commonly used protein structure visualization software (for example, PyMol, Chimera and other software can be used).
  • the structural information of Cas12i2 refers to PDB: 6LTU, 6LTR, 6LU0, 6LTP).
  • Cas12i2 effector protein was expressed in human 293T cells by pCAG-2A-eGFP vector.
  • the DNA encoding the Cas12i2 protein was inserted between XmaI and NheI.
  • a vector expressing Cas12i2crRNA in 293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-i2-crRNA backbone.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • the coding sequences of the crRNA spacers for CCR5-3 and RNF2-7 are shown in SEQ ID NO: 60 and 61, respectively.
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM Gibco
  • Gibco penicillin-streptomycin
  • Gibco fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • 600ng of a plasmid encoding Cas12i2 protein and 300ng of a plasmid encoding crRNA were transfected into each 24-well-cell culture dish.
  • HEK293T cells to be subjected to fluorescence-activated cell sorting were digested with trypsin-EDTA (0.05%) (Gibco).
  • Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP channel.
  • FACS-sorted GFP-positive HEK293T cells were lysed with buffer L and incubated at 55 °C for 3 h, then at 95 °C for 10 min.
  • dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers.
  • target loci are directly amplified by barcoded PCR using cell lysates as templates.
  • PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of indels was analyzed using CRISPResso2 software by calculating the ratio of reads containing indels or indels. Reads with an amount less than 0.05% of complete reads were discarded.
  • Figure 4 and Table 4 summarize the gene editing efficiencies of Cas12i2 mutants in this example and wild-type Cas12i2 at two genomic loci: CCR5-3 and RNF2-7 in 293T cells.
  • seven mutants G116R, E117R, T159R, S161R, E319R, E343R, and D958R were able to effectively improve gene editing efficiency (at least at one genomic locus).
  • D958R showed excellent gene editing efficiency at both genomic loci.
  • Example 5 Combine some of the Cas12i2 engineered amino acid mutations obtained in Examples 1-4 to improve gene editing efficiency, and verify their gene editing efficiency.
  • the combination of mutants is constructed by splitting the DNA of the Cas12i2 protein into multiple segments and using PCR and Gibson clone.
  • the location of the mutant is determined by analyzing the structural information of Cas12i2 using commonly used protein structure visualization software (for example, software such as PyMol and Chimera can be used).
  • the structural information of Cas12i2 refers to PDB: 6LTU, 6LTR, 6LU0, 6LTP).
  • Cas12i2 effector protein was expressed in human 293T cells by pCAG-2A-eGFP vector.
  • the DNA encoding the Cas12i2 protein was inserted between XmaI and NheI.
  • a vector expressing Cas12i2crRNA in 293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-i2-crRNA backbone.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM Gibco
  • Gibco penicillin-streptomycin
  • Gibco fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • 600ng of a plasmid encoding Cas12i2 protein and 300ng of a plasmid encoding crRNA were transfected into each 24-well-cell culture dish.
  • HEK293T cells to be subjected to fluorescence-activated cell sorting were digested with trypsin-EDTA (0.05%) (Gibco).
  • Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP channel.
  • FACS-sorted GFP-positive HEK293T cells were lysed with buffer L and incubated at 55 °C for 3 h, then at 95 °C for 10 min.
  • dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers.
  • target loci are directly amplified by barcoded PCR using cell lysates as templates.
  • PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of indels was analyzed using CRISPResso2 software by calculating the ratio of reads containing indels or indels. Reads with an amount less than 0.05% of complete reads were discarded.
  • mutants As shown in Figure 5 and Table 5, by comparing these mutants and wild-type Cas12i in 293T cells, they act on 5 genomic sites: CCR5-3, CCR5-5, CD34-8, CD34-9, RNF2-14
  • mutants With further improved efficiency can be obtained.
  • the mutant E176R+K238R+T447R+E563R+N164Y+E323R+D362R
  • the coding sequences of the used crRNA spacers against CD34-8, CD34-9 and RNF2-14 are shown in SEQ ID NO: 63, 64 and 65, respectively.
  • Example 5 Summary of the results (gene editing efficiency) of Example 5 (only the gene editing efficiency data embodied at RNF2-14 is taken as an example)
  • *L439(L+G) means that a glycine is inserted after amino acid No. 439; in the context of this specification and the drawings, it is sometimes expressed as 439G.
  • T7 endonuclease 1 T7E1
  • Example 6 Comparison of CasXX and conventional gene editing tools to verify its gene editing efficiency.
  • Cas effector protein was expressed in human 293T cells by pCAG-2A-eGFP vector.
  • the DNA encoding the Cas protein was inserted between XmaI and NheI.
  • sgRNAs or crRNAs expressing AsCas12a, BhCas12b v4, SpCas9, SaCas9, SaCas9-KKH and Cas12i2 in 293T were constructed by ligating annealed oligonucleotides containing target sequences into BasI-digested pUC19-U6-i2-crRNA backbone Carrier.
  • the nucleotide sequence encoding the DR of CasXX is SEQ ID NO:59.
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM Gibco
  • Gibco penicillin-streptomycin
  • Gibco fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • 600ng of a plasmid encoding Cas12i2 protein and 300ng of a plasmid encoding crRNA were transfected into each 24-well-cell culture dish.
  • HEK293T cells to be subjected to fluorescence-activated cell sorting were digested with trypsin-EDTA (0.05%) (Gibco).
  • Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP channel.
  • FACS-sorted GFP-positive HEK293T cells were lysed with buffer L and incubated at 55 °C for 3 h, then at 95 °C for 10 min.
  • dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers.
  • target loci are directly amplified by barcoded PCR using cell lysates as templates.
  • PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of indels was analyzed using CRISPResso2 software by calculating the ratio of reads containing indels or indels. Reads with an amount less than 0.05% of complete reads were discarded.
  • the pCAG-2A-eGFP vector encoding CasXX and the pUC19-U6-i2-crRNA vector encoding crRNA were transfected into the mouse Hepa1-6 liver cancer cell line by lipofection. Among them, corresponding crRNAs were designed for 65 endogenous gene loci.
  • the designed spacer sequence is 20 nucleotides.
  • the indel frequency is obtained by PCR amplification and sequencing, which is similar to the analysis method of exogenous gene editing.
  • CasXX exhibited powerful gene editing ability on 65 endogenous gene loci of the mouse Hepa1-6 cell line, with an average gene editing efficiency of more than 60%.
  • Example 7 Gene editing at genomic loci containing different PAMs using CasXX.
  • the DNA sequence encoding CasXX was loaded into the pCAG-2A-EGFP plasmid, thereby constructing the expression CasXX plasmid.
  • a vector for expression of crRNA in HEK293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-crRNA backbone. Among them, 64 different crRNAs were designed to target 64 human endogenous sites.
  • the PAM sequences contained in these 64 sites cover all NNNN combinations: NTTN, NTAN, NTCN, NTGN, NATN, NAAN, NACN, NAGN, NCTN, NCAN, NCCN, NCGN, NGTN, NGAN, NGCN, NGGN.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • the designed spacer sequence is 20 nucleotides.
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • 600 ng of a plasmid encoding a Cas protein and 300 ng of a plasmid encoding a crRNA were transfected into cells cultured in a 24-well cell culture dish. 72h after transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco), and then subjected to GFP fluorescence-activated cell sorting (FACS).
  • FACS-sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55 °C for 3 h, then at 95 °C for 10 min.
  • dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers.
  • target loci are directly amplified by barcoded PCR using cell lysates as templates.
  • PCR products were purified and pooled into several libraries for high-throughput sequencing.
  • the frequency (%) of indels was analyzed using CRISPResso2 software by calculating the ratio of reads containing indels or indels. Reads with an amount less than 0.05% of complete reads were discarded.
  • the experimental results are shown in Figure 7.
  • the target sites of CasXX with NTTN, NTAN, NTCN, NTGN, NATN, NAAN, NCTN, NCAN, and NGTN at the 5' end of these PAMs all exhibited high gene editing efficiency.
  • the average gene editing efficiency exceeds 40%.
  • Example 8 In vitro cleavage of double-stranded DNA containing different PAMs using CasXX
  • the BPK2014 prokaryotic expression plasmid was transformed into Escherichia coli strain BL21( ⁇ DE3) (TransGen Biotech), and the transformed bacterial liquid was spread on solid LB containing chloramphenicol. Three clones were picked into 5 ml liquid LB and grown overnight. Then the bacteria were transferred to 3L liquid LB to continue culturing until the OD600 reached 0.6-0.8. It was then induced with IPTG (0.5 mM) for 20 hours at 16°C. Bacteria expressing Cas12i were harvested by ultracentrifugation, resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl) and disrupted by sonication.
  • lysis buffer 50 mM Tris-HCl, pH 7.5, 300 mM NaCl
  • the Cas12i protein in the supernatant was first purified with a Ni column. Briefly, after incubation with the supernatant, the Ni column was sequentially washed with lysis buffer supplemented with 0 mM, 20 mM and 50 mM imidazole. Then, Cas12i protein was eluted by lysis buffer supplemented with 500 mM imidazole. The collected samples were then loaded into an ion exchange column (CM Sepharose Fast Flow, GE). Wild-type Cas12i2 and CasXX proteins were eluted with storage buffer (20 mM Tris-HCl, 300 mM NaCl, 1 mM TCEP, 10% glycerol, pH7.5). Proteins were filter sterilized and stored at -80 °C.
  • the nucleotide sequence encoding DR is SEQ ID NO:59. Synthesize the oligonucleotide containing the T7 promoter sequence (named T7-F) and the oligonucleotide containing the complementary sequence of crRNA and T7 promoter (named T7-12i-crRNA-R), and in 1xNEBufferTM2 (NEB) Medium annealing. The sequences of these oligonucleotides are listed in Table 6. The annealed product was used as a template to produce crRNA by HiScribeTM T7 Quick High Yield RNA Synthesis Kit (NEB). use RNA Cleanup Kit (NEB) purifies transcribed crRNA.
  • targets containing the same protospacer but different PAMs were first cloned into EcoR1 and HindIII treated pUC19 (with penicillin resistance). Target sequences with 5' PAMs are listed in Table 7.
  • the pUC19 plasmid carrying the target was then linearized by SacI and purified using DNA Clean & Concentrator (Zymo Research).
  • 400 nM Cas12i protein was first incubated with 2 ⁇ M crRNA at 37 °C for 15 min.
  • the wild-type Cas12i2 protein only partially cuts double-stranded DNA containing 5'-NTTN-3'PAM, but has almost no cutting activity on the rest of the PAM.
  • CasXX protein showed high cutting efficiency for double-stranded DNA containing NTTN, NTAN, NTCN, NTGN, NATN, NAAN, NACN, NCTN, NCAN, NGTN, and NGAN PAM.
  • CasXX can almost completely cleave double-stranded DNA containing NTTN, NTAN, NTCN, NATN, NAAN, NACN, NCTN, NCAN, NGTN PAM. Therefore, the engineered Cas12i nuclease provided by the present invention can be used for more extensive gene editing or treatment.
  • a vector expressing Cas protein crRNA in 293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-crRNA backbone.
  • the crRNA comprises a spacer sequence (spacer; SEQ ID NO: 77) capable of targeting the endogenous site of EMX1-7.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • 600ng of a plasmid encoding a Cas protein 300ng of a plasmid encoding a crRNA
  • 10pmol of an annealed double-stranded DNA tag see Table 8 for the sequence
  • the cells were digested with trypsin-EDTA (0.05%) (Gibco), and then subjected to GFP fluorescence-activated cell sorting (FACS). The cells that successfully express the Cas enzyme were selected.
  • the MicroElute Genomic DNA Kit (Omega) kit was used to extract the genome of FACS-sorted GFP-positive 293T cells. The purified genome was quantified using Qubit. Using the Covaris S220 instrument, the genome was fragmented to about 500bp according to the program recommended by the instrument. Then use the VAHTS Universal Pro DNA Library Prep Kit for Illumina (Vazyme) kit for DNA library construction.
  • the library construction process refers to references (Tsai, SQ et al. "GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases," Nat Biotechnol.2015; 33(2): 187-197; its content incorporated herein by reference in its entirety).
  • the library products were subjected to high-throughput sequencing. Analyze sequencing results to find potential off-target sites. We systematically analyzed the off-target effect of CasXX at the EMX1-7 site, and the specific analysis results are shown in Figure 10.
  • the first line of sequence is the reference target sequence, and the following sequences represent the target sequence and off-target sequence and the number of reads enriched by double-stranded DNA tags in the target sequence and off-target sequence, respectively.
  • Example 10 On the basis of the CasXX sequence (SEQ ID NO: 8), a new amino acid mutation is introduced to check the characteristics of the mutant's specificity improvement (two targeting sites are selected for mutant screening experiments, namely EMX1-7 locus and RNF2-1 locus)
  • CasXX including N164Y+E176R+K238R+E323R+D362R+T447R+E563R mutation based on SEQ ID NO: 1
  • SEQ ID NO: 8 Based on the sequence of CasXX (SEQ ID NO: 8), we used primers with mutated bases to perform PCR on the DNA sequence of CasXX, using The HiFi DNA Assembly Master Mix (NEB) kit loaded the purified PCR product into the pCAG-2A-EGFP plasmid, thereby constructing the CasXX plasmid expressing the introduced point mutation.
  • NEB HiFi DNA Assembly Master Mix
  • CasXX-HF-1 a total of 26 CasXX sequence-based mutants containing single amino acid mutations, named CasXX-HF-1 to HF-26, respectively.
  • the specific mutation methods are shown in Table 9.
  • CasXX-HF-26 restores the N164Y point mutation in CasXX to the original amino acid N of wild-type Cas12i2, that is, Y164N (that is, the mutation N164Y is deleted).
  • a vector expressing Cas protein crRNA in 293T was constructed by ligating the annealed oligonucleotide containing the target sequence into the pUC19-U6-crRNA backbone digested with BasI (NEB) using T4ligase (NEB).
  • the crRNA was designed to target EMX1-7 sites.
  • the specific sequence is shown in Table 10.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • serial number crRNA target site name spacer coding sequence SEQ ID NO: 27 EMX1-7 TGGTTGCCCACCCTAGTCAT
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • 600 ng of a plasmid encoding a Cas protein and 300 ng of a plasmid encoding a crRNA were transfected into cells cultured in a 24-well cell culture dish. 72h after transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco), and then subjected to GFP fluorescence-activated cell sorting (FACS).
  • FACS-sorted GFP-positive HEK293T cells were lysed with 40 ⁇ L of buffer L (bimake), incubated at 55°C for 3 hours, and then incubated at 95°C for 10 minutes. Containing target sites in different genomic loci using primer pairs for EMX1-7 target site, EMX1-7 off-target site 1, EMX1-7 off-target site 2, and EMX1-7 off-target site 3 ( Figure 10)
  • the dsDNA fragments of the target or off-target sites were amplified by PCR, and the sequences of the target sites or off-target sites are shown in Table 11. Afterwards, using 10 ⁇ L of the PCR product, a re-annealing procedure was performed to form heteroduplex dsDNA.
  • a vector expressing Cas protein crRNA in HEK293T was constructed by ligating the annealed oligonucleotide containing the target sequence into the pUC19-U6-crRNA backbone digested with BasI (NEB) using T4ligase (NEB).
  • the final crRNAs all target the same RNF2-1 site, but the spacer sequences are different.
  • the specific sequences encoding these spacers are shown in Table 13.
  • RNF2-1-FM means that the spacer sequence in crRNA completely matches the RNF2-1 site.
  • RNF2-1-Mis-1/2 means that the spacer sequence in crRNA does not match the RNF2-1 site at the 1st and 2nd base positions, and matches at the remaining positions.
  • RNF2-1-Mis-5/6 means that the spacer sequence in the crRNA does not match the 5th and 6th base positions of the RNF2-1 site, and matches at the remaining positions.
  • RNF2-1-Mis-17/1 means that the spacer sequence in the crRNA does not match the 17th and 18th base positions of the RNF2-1 site, and matches at the remaining positions.
  • RNF2-1-Mis-19/20 means that the spacer sequence in the crRNA does not match the 19th and 20th base positions of the RNF2-1 site, and matches at the remaining positions.
  • the purpose of setting base mismatches is to simulate off-target effects.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • crRNA name spacer coding sequence 32 crRNA-RNF2-1-FM TACAGGAGGCAATAACAGAT 33 crRNA-RNF2-1-Mis-1/2 GCCAGGAGGCAATAACAGAT 34 crRNA-RNF2-1-Mis-5/6 TACATTAGGCAATAACAGAT 35 crRNA-RNF2-1-Mis-17/18 TACAGGAGGCAATAACCTAT 36 crRNA-RNF2-1-Mis-19/20 TACAGGAGGCAATAACAGCG
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • 600ng of a plasmid encoding a Cas protein and 300ng of a plasmid encoding a crRNA were transfected into cells cultured in a 24-well cell culture dish. 72h after transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco), and then subjected to GFP fluorescence-activated cell sorting (FACS). The cells that successfully express the Cas enzyme were selected.
  • FACS-sorted GFP-positive HEK293T cells were lysed with 40 ⁇ L of buffer L (bimake), incubated at 55°C for 3 hours, and then incubated at 95°C for 10 minutes.
  • the dsDNA fragment at the RNF2-1 site was amplified by PCR using the RNF2-1 primer pair. Afterwards, using 10 ⁇ L of the PCR product, a re-annealing procedure was performed to form heteroduplex dsDNA. Then, the mixture was treated with 1/10 volume of NEBuffer TM 2.1 and 0.2 ⁇ L of T7 endonuclease I (NEB) at 37° C. for 50 minutes. Digestion products were analyzed by ⁇ 2.5% agarose gel electrophoresis.
  • the indel ratio (Indel, %) was calculated according to the gray value of the band.
  • the ratio of indels at the target site for each Cas12i mutant when using different crRNAs is shown in Table 14, corresponding to FIG. 12 .
  • Experimental results show that R857A, N861A, K807A, N848A, R715A, R719A, K394A, H357A, K844A these single point mutants based on CasXX sequence (corresponding to the engineered Cas12i nuclease of SEQ ID NOs: 14-22, see Table 9)
  • Even when using simulated off-target crRNA-RNF2-1-Mis-1/2, crRNA-RNF2-1-Mis-5/6, crRNA-RNF2-1-Mis-17/18 and crRNA-RNF2-1-Mis- 19/20 can still effectively reduce the proportion of indels.
  • we defined the decrease of the indel rate at the off-target site and the substantially unchanged (or increased) editing efficiency at the target site as
  • Table 14 Editing efficiency of different CasXX-HF mutants at the RNF2-1 site using crRNA targeting the target sequence and simulating the off-target spacer sequence
  • Embodiment 11 The mutant that introduces new amino acid mutation on the basis of CasXX sequence
  • mutants with new amino acid mutations introduced on the basis of the CasXX sequence those mutants with improved specificity were screened in the fluorescent reporter system.
  • the difference between this example and Example 10 is that the analysis means reflecting the editing efficiency is obtained by agarose gel Electrophoresis was changed to fluorescence.
  • a vector expressing Cas protein crRNA in HEK293T was constructed by ligating the annealed oligonucleotide containing the target sequence into the pUC19-U6-crRNA backbone digested with BasI (NEB) using T4ligase (NEB).
  • the final crRNAs all target the same mCherry site, but the spacer sequences are different.
  • the specific sequence encoding the spacer is shown in Table 15. Among them, mCherry-FM means that the spacer sequence in the crRNA completely matches the mCherry site.
  • crRNA-mCherry-Mis-1/2 means that the spacer sequence in the crRNA does not match the crRNA-mCherry site at the 1st and 2nd base positions, and matches at the remaining positions.
  • crRNA-mCherry-Mis-5/6 means that the spacer sequence in the crRNA does not match the 5th and 6th base positions of the crRNA-mCherry site, and matches at the remaining positions.
  • crRNA-mCherry-Mis-19/20 means that the spacer sequence in the crRNA does not match the crRNA-mCherry site at the 19th and 20th base positions, and matches at the remaining positions.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • Table 15 Spacer coding sequence targeting mCherry and simulating off-target
  • crRNA target site name Spacer coding sequence 37 crRNA-mCherry-FM ACCTTGTAGATGAACTCGCC 38 crRNA-mCherry-Mis-1/2 CACTTGTAGATGAACTCGCC 39 crRNA-mCherry-Mis-5/6 ACCTGTTAGATGAACTCGCC 40 crRNA-mCherry-Mis-19/20 ACCTTGTAGATGAACTCGAA
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM Gibco
  • Gibco penicillin-streptomycin
  • Gibco fetal bovine serum
  • 600 ng of a plasmid encoding a Cas protein, 300 ng of a plasmid encoding a crRNA, and 100 ng of a plasmid encoding mCherry were transfected into cells cultured in a 24-well cell culture dish.
  • Example 12 On the basis of the CasXX sequence, amino acid combination mutations are introduced, and endogenous gene (combination) sites are screened for mutants with improved specificity
  • a vector expressing Cas protein crRNA in 293T was constructed by ligating the annealed oligonucleotide containing the target sequence into the pUC19-U6-crRNA backbone digested with BasI (NEB) using T4ligase (NEB).
  • crRNAs were designed as described in Example 10 to target EMX1-7 or RNF2-1, or to mimic RNF2-1 off-targets. See Tables 10 and 13 for the Spacer coding sequence.
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the cell density reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of the plasmid encoding the Cas protein and 300ng of the plasmid encoding the crRNA were transfected into cells cultured in a 24-well cell culture dish. 72 h after transfection, cells were digested with trypsin-EDTA (0.05%) (Gibco), and then subjected to fluorescence-activated cell sorting (FACS). The cells that successfully express the Cas enzyme were selected.
  • FACS-sorted GFP-positive HEK293T cells were lysed with 40 ⁇ L of buffer L (bimake), incubated at 55°C for 3 hours, and then incubated at 95°C for 10 minutes.
  • the corresponding primers described in Example 10 were used to perform PCR amplification on dsDNA fragments containing target sites or off-target sites in different genomic loci.
  • the sequences of the target sites or off-target sites are shown in Tables 11 and 13. Afterwards, using 10 ⁇ L of the PCR product, a re-annealing procedure was performed to form heteroduplex dsDNA.
  • the mixture was treated with 1/10 volume of NEBuffer TM 2.1 and 0.2 ⁇ L of T7 endonuclease I (NEB) at 37° C. for 50 minutes. Digestion products were analyzed by ⁇ 2.5% agarose gel electrophoresis. The indel ratio (Indel, %) was calculated according to the gray value of the band.
  • R857A, R719A, K394A, K844A, R719A/K844A and R857A/K844A mutants based on CasXX sequences were used to mimic off-target crRNA-RNF2-1-Mis-1/2, crRNA-RNF2-1-Mis-5 /6, crRNA-RNF2-1-Mis-17/18 and crRNA-RNF2-1-Mis-19/20 can effectively reduce the ratio of indels without sacrificing the efficiency at the target site, see Table 18, corresponding Figure 16.
  • mutants shown in SEQ ID NO: 23 and SEQ ID NO: 24 in this description (corresponding to the mutants marked with sequence numbers in Table 16) have significantly higher specificity.
  • Example 13 Detection of off-target effects of CasXX and CasXX+K394A mutant using GUIDE-Seq.
  • the DNA sequences encoding CasXX (SEQ ID NO: 8) and CasXX+K394A mutant (SEQ ID NO: 20) were loaded into the pCAG-2A-EGFP plasmid, thereby constructing a plasmid expressing CasXX or (CasXX+K394A).
  • a vector expressing Cas12i protein crRNA in HEK293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-crRNA backbone.
  • the crRNA is designed to contain a spacer sequence that can target the endogenous site of CD34-7.
  • the nucleotide sequence encoding DR is SEQ ID NO:59.
  • the nucleotide sequence encoding spacer comprises SEQ ID NO:78.
  • HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the cell density reached 70%.
  • DMEM fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • the MicroElute Genomic DNA Kit (Omega) kit was used to extract genomes from FACS-sorted GFP-positive HEK293T cells. The purified genome was quantified using Qubit. Using the Covaris S220 instrument, the genome was fragmented to about 500bp according to the program recommended by the instrument. Then use the VAHTS Universal Pro DNA Library Prep Kit for Illumina (Vazyme) kit for DNA library construction.
  • the library construction process refers to references (Tsai, SQ et al. "GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases," Nat Biotechnol. 2015; 33(2): 187-197).
  • the library products were subjected to high-throughput sequencing. Analyze sequencing results to find potential off-target sites.
  • the first line of sequence is the reference target sequence, and the following sequences represent the target sequence and off-target sequence and the number of reads enriched by double-stranded DNA tags in the target sequence and off-target sequence, respectively.
  • Figure 17 shows that CasXX has an off-target effect at the CD34-7 site, but the CasXX+K394A mutant has no off-target effect at this site. Therefore, the additional introduction of the K394A point mutation on the basis of the CasXX sequence can greatly improve the fidelity of CasXX, making CasXX highly specific.

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Abstract

提供了一种工程化的Cas12i核酸酶；其包含一种或多种以下基于参比Cas12i核酸酶的突变：(1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸；和/或(2)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸；和/或(3)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸；和/或(4)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为带正电的氨基酸；和/或(5)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸。该工程化的Cas12i核酸酶较参比Cas12i核酸酶具有显著提高的基因编辑效率和/或显著降低的脱靶现象。

Description

工程化的Cas12i核酸酶、效应蛋白及其用途
优先权信息
本申请要求：于2021年11月15日提交的中国专利申请CN202111347952.7、于2021年5月27日提交的中国专利申请CN202110581290.3、以及于2021年5月27日提交的国际申请PCT/CN2021/096477的优先权，它们的全部内容通过整体引用并入本文。同时提交的序列表文件
下列ASCII码文本文件的全部内容通过整体引用并入本文：计算机可读格式(CRF)的序列表(FF00614PCT-NSequence6thVersion-20220525.txt，日期：2022年5月25日，大小：218kb)
技术领域
本申请属于生物技术领域。更具体地说，本申请涉及具有提高的催化活性(例如基因编辑活性)的Cas12i核酸酶、效应蛋白及其用途。
背景技术
基因组编辑是在基因组研究中的重要且有用的技术。有多个系统可用于基因组编辑，包括成簇规律间隔短回文重复序列(CRISPR)-Cas系统、转录激活子样效应因子核酸酶(TALEN)系统以及锌指核酸酶(ZFN)系统。
CRISPR-Cas系统是一种高效且具有成本效益的基因组编辑技术，可广泛应用于从酵母、植物到斑马鱼和人类的一系列真核生物中(参见综述：Van der Oost 2013，Science 339：768-770，以及Charpentier and Doudna，2013，Nature 495：50-51)。CRISPR-Cas系统通过结合Cas12i效应蛋白和CRISPR RNA(crRNA)在古细菌和细菌中提供适应性免疫。迄今为止，基于该系统的突出的功能上和进化上的模块性，已经对包括六型(I-VI型)两类(第1类和第2类)的CRISPR-Cas系统进行了表征。在第2类CRISPR-Cas系统中，II型Cas9系统和V型-A/B/E/J Cas12a/Cas12b/Cas12e/Cas12j系统已被利用来进行基因组编辑，并为生物医学研究提供了广阔的前景。
但是，当前的CRISPR-Cas系统具有多种局限性，包括有限的基因编辑效率。因此， 需要改进方法和系统以进行有效的跨多基因座的基因组编辑。
发明概述
本申请提供了下述技术方案：
1.一种工程化的Cas12i核酸酶；其包含一种或多种基于参比Cas12i核酸酶的突变，所述突变选自：
(1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸；
(2)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸
(3)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸
(4)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为带正电的氨基酸；和
(5)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸；
优选地，所述参比Cas12i核酸酶为氨基酸序列为SEQ ID NO：1的野生型Cas12i2核酸酶。
2.如项1所述的工程化的Cas12i核酸酶，其中，所述与PAM相互作用的一个或多个氨基酸是与PAM在三维结构上距离在9埃以内的氨基酸；优选，所述与PAM相互作用的一个或多个氨基酸是下述位置的一个或多个氨基酸：176、178、226、227、229、237、238、264、447和563，
进一步优选，所述与PAM相互作用的一个或多个氨基酸是下述一个或多个氨基酸：E176、E178、Y226、A227、N229、E237、K238、K264、T447和E563，
进一步优选，所述与PAM相互作用的一个或多个氨基酸是下述一个或多个氨基酸：E176、K238、T447和E563，
其中，所述氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
3.如项1或2所述的工程化的Cas12i核酸酶，其中，所述带正电的氨基酸是R，K或H。
4.如项1-3中任一项所述的工程化的Cas12i核酸酶，其中，所述将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸是指如下替换中的一种 或多种：E176R、K238R、T447R和E563R；
优选，所述工程化的Cas12i核酸酶包含下述任何一种突变或突变组合：(1)E563R；(2)E176R、T447R、E176R和E563R；(3)K238R和E563R；(4)E176R、K238R和T447R；(5)E176R、K238R和E563R；(6)E176R、T447R和E563R；和(7)E176R、K238R、T447R和E563R；
其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
5.如项1～4中任一项所述的工程化的Cas12i核酸酶，其中，所述参与打开DNA双链的一个或多个氨基酸是与PAM中相对于靶标链的3’端最后一个碱基对相互作用的氨基酸；
优选，所述参与打开DNA双链的一个或多个氨基酸是下述位置的一个或多个氨基酸：163和164；
进一步优选，所述参与打开DNA双链的一个或多个氨基酸是下述一个或多个氨基酸：Q163和N164；进一步优选，所述参与打开DNA双链的氨基酸为N164；
其中，所述氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
6.如项1-5中任一项所述的工程化的Cas12i核酸酶，其中，所述参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸，所述带芳香环的氨基酸是F、Y或W，优选，所述带芳香环的氨基酸为F或Y。
7.如项1-6中任一项所述的工程化的Cas12i核酸酶，其中，所述将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸的替换为带芳香环的氨基酸包括以下一个或多个替换：Q163F、Q163Y、Q163W、和N164F；优选，所述Cas12i核酸酶包含N164Y或N164F突变；进一步优选，所述工程化的Cas12i核酸酶包含N164Y。
8.如项1～7中任一项所述的工程化的Cas12i核酸酶，其中，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是在三维结构上与单链DNA底物距离在9埃以内的氨基酸；
优选，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是下述位置的一个或多个氨基酸：323、327、355、359、360、361、362、388、390、391、392、393、414、417、418、421、424、425、650、652、653、696、705、708、709、751、752、755、840、848、851、856、885、897、925、926、928、929、932、1022。
进一步优选，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是下述一个或多个氨基酸：E323、L327、V355、G359、G360、K361、D362、L388、N390、N391、F392、K393、Q414、L417、L418、K421、Q424、Q425、S650、E652、 G653、I696、K705、K708、E709、L751、S752、E755、N840、N848、S851、A856、Q885、M897、N925、I926、T928、G929、Y932、A1022；
进一步优选，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是是下述一个或多个氨基酸：E323、D362、Q425、N925、I926、N391、Q424和G929；
其中，所述氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
9.如项1-8中任一项所述的工程化的Cas12i核酸酶，其中，将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸包括替换为R或K，优选，所述带正电的该氨基酸为R。
10.如项1-9中任一项所述的工程化的Cas12i核酸酶，其中，将参比Cas12i核酸酶位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸是指包括如下替换中的一种或多种：E323R、D362R、N391R、Q424R、Q425R、N925R、I926R和G929R；
优选，所述工程化的Cas12i核酸酶包含下述任何一种突变或突变组合：(1)E323R；(2)D362R；(3)Q425R；(4)N925R；(5)I926R；(6)E323R和D362R；(7)E323R和Q425R；(8)E323R和I926R；(9)Q425R和I926R；(10)D362R和I926R；(11)N925R和I926R；(12)E323R、D362R和Q425R；(13)E323R、D362R和I926R；(14)E323R、Q425R和I926R；(15)D362R、N925R和I926R；和(16)E323R、D362R、Q425R和I926R；
其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
11.如项1～10中任一项所述的工程化的Cas12i核酸酶，其中，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是与DNA-RNA双螺旋在三维结构上距离在9埃以内的氨基酸；
优选，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是下述位置的一个或多个氨基酸：116、117、156、159、160、161、247、293、294、297、301、305、306、308、312、313、316、319、320、343、348、349、427、433、438、441、442、679、683、691、782、783、797、800、852、853、855、861、865、957、958；
进一步优选，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是下述一个或多个氨基酸：G116、E117、A156、T159、S161、T301、I305、K306、T308、N312、F313、D427、K433、V438、N441、Q442、M852、L855、N861、Q865、E160、Q316、E319、Q320、E247、E343、E348、E349、N679、E683、E691、D782、E783、E797、E800、D853、S957、D958、G293、E294和N297；G116、E117、A156、T159、E160、 S161、E247、G293、E294、N297、1301、1305、K306、T308、N312、F313、Q316、E319、Q320、E343、E348、E349、D427、K433、V438、N441、Q442、N679、E683、E691、D782、E783、E797、E800、M852、D853、L855、N861、Q865、S957、D958；
进一步优选，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是下述一个或多个氨基酸：G116、E117、T159、S161、E319、E343和D958；其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
12.如项1-11中任一项所述的工程化的Cas12i核酸酶，其中，将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为带正电的氨基酸包括替换为R或K，优选，所述带正电的氨基酸为R。
13.如项1-12中任一项所述的工程化的Cas12i核酸酶，其中，将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为带正电的氨基酸包括替换为以下的一种或多种：G116R、E117R、T159R、S161R、E319R、E343R和D958R；
优选，所述工程化的Cas12i核酸酶包含D958R替换；
其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
14.如项1～13中任一项所述的工程化的Cas12i核酸酶，所述与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸选自如下的一个或多个位置的氨基酸：357、394、715、719、807、844、848、857、861，即下述一个或多个氨基酸：H357、K394、R715、R719、K807、K844、N848、R857、R861；
其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
15.如项1-14中任一项所述的工程化的Cas12i核酸酶，其中，将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸包括替换为丙氨酸(A)。
16.如项1-15中任一项所述的工程化的Cas12i核酸酶，其包含选自下组的一个或多个突变：H357A、K394A、R715A、R719A、K807A、K844A、N848A、R857A、R861A；
优选，包含选自下组的一个或多个突变：K394A、R719A、K844A、R857A；
其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
17.如项1-16中任一项所述的工程化的Cas12i核酸酶，其包含R719A和K844A氨基酸取代，或包含R857A和K844A氨基酸取代；其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
18.如项1～17中任一项所述的工程化的Cas12i核酸酶，其还包含一个或多个柔性区突变，所述突变增加了参比Cas12i核酸酶中的柔性区的柔性，所述柔性区选自氨基酸残 基439-443或氨基酸残基925-929；
优选，所述柔性区突变位于439和/或926位点；
进一步优选，所述柔性区突变是L439和/或I926的突变；
其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
19.如项18所述的工程化的Cas12i核酸酶，其中，所述一个或多个柔性区突变为：将该柔性区氨基酸替换为G、和/或在其后插入一个或两个G；
优选，所述一个或多个柔性区突变包含I926G、L439(L+G)或L439(L+GG)；
进一步优选，所述一个或多个柔性区突变包含L439(L+G)或L439(L+GG)；
其中，所述氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
20.一种工程化的Cas12i核酸酶；
所述工程化的Cas12i核酸酶包含任何以下一组或多组突变：
(1)E563R；(2)E176R和T447R；(3)E176R和E563R；(4)K238R和E563R；(5)E176R、K238R和T447R；(6)E176R、T447R和E563R；(7)E176R、K238R和E563R；(8)E176R、K238R、T447R和E563R；(9)N164Y；(10)N164F；(11)E323R；(12)D362R；(13)Q425R；(14)N925R；(15)I926R；(16)D958R；(17)E323R和D362R；(18)E323R和Q425R；(19)E323R和I926R；(20)Q425R和I926R；(21)D362R和I926R；(22)N925R和I926R；(23)E323R、D362R和Q425R；(24)E323R、D362R和I926R；(25)E323R、Q425R和I926R；(26)D362R、N925R和I926R；(27)E323R、D362R、Q425R和I926R；(28)D362R和I926G；(29)N925R和I926G；(30)D362R、N925R和I926G；(31)I926R和L439(L+G)；(32)I926R和L439(L+GG)；(33)E323R、D362R和I926G；(34)R719A和K844A；和(35)R857A和K844A；
优选，所述工程化的Cas12i核酸酶包含任何以下一组或多组突变：(1)E176R、K238R、T447R和E563R；(2)N164Y；(3)I926R；(4)E323R和D362R；(4)I926G；(5)I926R和L439(L+G)；(6)I926R和L439(L+GG)；和(7)D958R；
其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
21.一种工程化的Cas12i核酸酶，所述工程化的Cas12i核酸酶包含以下任一组突变：(1)E176R、K238R、T447R、E563R和N164Y；(2)E176R、K238R、T447R、E563R和I926R；(3)N164Y、E323R和D362R；(4)E176R、K238R、T447R、E563R、E323R和D362R；(5)N164Y和I926R；(6)E176R、K238R、T447R、E563R、N164Y和I926R；(7)E176R、K238R、T447R、E563R、N164Y、E323R和D362R；(8)E176R、K238R、T447R、E563R、N164Y、I926R、E323R和D362R；(9)E176R、K238R、T447R、E563R、 N164Y、E323R、D362R和I926G；(10)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G和L439(L+GG)；(11)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G和L439(L+G)；(12)E176R、K238R、T447R、E563R、N164Y和D958R；(13)E176R、K238R、T447R、E563R、I926R和D958R；(14)E176R、K238R、T447R、E563R、E323R、D362R和D958R；(15)N164Y、I926R和D958R；(16)N164Y、E323R、D362R和D958R；(17)E176R、K238R、T447R、E563R、N164Y、I926R和D958R；(18)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和D958R；(19)E176R、K238R、T447R、E563R、N164Y、I926R、E323R、D362R和D958R；(20)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G和D958R；(21)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G、L439(L+GG)和D958R；(22)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G、L439(L+G)和D958R；(23)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和R857A；(24)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和N861A；(25)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和K807A；(26)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和N848A；(27)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和R715A；(28)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和R719A；(29)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和K394A；(30)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和H357A；(31)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和K844A；(32)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、R719A和K844A；或(33)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、R857A和K844A；其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
22.如项1～21中任一项所述的工程化的Cas12i核酸酶，其包含如SEQ ID NOs：2～24中任一项所示氨基酸序列的工程化Cas12i核酸酶，或与如SEQ ID NOs：2～24中任一序列所示的氨基酸序列具有至少80％同一性的氨基酸序列。
23.一种工程化的Cas12i效应蛋白，其包含项1～22中任一项所述的工程化的Cas12i核酸酶或其功能衍生物；
任选的，所述工程化的Cas12i核酸酶或其功能衍生物具有酶活性，或者所述工程化的Cas12i核酸酶或其功能衍生物为酶失活突变体。
24.如项23所述的工程化的Cas12i效应蛋白，其中所述Cas12i效应蛋白能够诱导DNA分子中的双链断裂或单链断裂。
25.如项23所述的工程化的Cas12i效应蛋白，其中所述工程化的Cas12i核酸酶或其功能衍生物为包含以下一种或多种突变的酶失活突变体：D599A、E833A、S883A、H884A、R900A和D1019A；其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
26.如项23～25中任一项所述的工程化的Cas12i效应蛋白，其还包含与所述工程化的Cas12i核酸酶或其功能衍生物融合的功能结构域。
27.如项26所述的工程化的Cas12i效应蛋白，其中所述功能结构域选自下组中的一个或多个：翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域、逆转录酶结构域、报告分子结构域和核酸酶结构域。
28.如项23-27中任一项所述的工程化的Cas12i效应蛋白，所述工程化的Cas12i效应蛋白包含：含有所述工程化的Cas12i核酸酶或其功能衍生物的N末端部分的第一多肽和含有所述工程化的Cas12i核酸酶或其功能衍生物的C末端部分的第二多肽，其中所述第一多肽和所述第二多肽能够在包含指导序列的指导RNA的存在下彼此缔合，以形成与靶核酸特异性结合的成簇规律间隔短回文重复序列(CRISPR)复合物，所述靶核酸包含与所述指导序列互补的靶序列；
优选，所述第一多肽包含项1-22中任一项所述的工程化的Cas12i核酸酶的N末端部分氨基酸残基1至X，所述第二多肽包含项1-22中任一项所述的工程化的Cas12i核酸酶的氨基酸残基X+1至所述Cas12i核酸酶的C末端；
可选的，所述第一多肽和所述第二多肽各自包含二聚化结构域；
可选的，所述第一二聚结构域和所述第二二聚结构域在诱导剂存在下彼此缔合。
29.一种工程化的CRISPR-Cas12i系统，包括：
(a)项1-28中任一项所述的工程化的Cas12i核酸酶、或项23-28中任一项所述的工程化的Cas12i效应蛋白；以及
(b)包含与靶序列互补的指导序列的指导RNA，或编码所述指导RNA的一种或多种核酸.
其中所述工程化的Cas12i核酸酶或工程化的Cas12i效应蛋白和所述指导RNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰；
优选，所述指导RNA是包含所述指导序列的crRNA；
进一步优选，所述工程化的CRISPR-Cas12i系统包括编码多个crRNA的前体指导RNA阵列(array)；
或者，优选所述工程化的Cas12i核酸酶或工程化的Cas12i效应蛋白是主编辑器，所述指导RNA是引导编辑指导RNA(pegRNA)。
30.如项29所述的工程化的CRISPR-Cas12i系统，其包含一种或多种编码所述工程化的Cas12i核酸酶或工程化的Cas12i效应蛋白的载体；
优选，所述一种或多种载体选自下组：逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关的载体和单纯疱疹载体；
进一步优选，所述一种或多种载体是腺相关病毒AAV载体；
进一步优选，所述AAV载体还编码所述指导RNA。
31.一种检测样品中靶核酸的方法，包括：
(a)使样品与项29中的工程化的CRISPR-Cas12i系统以及加标签的检测核酸接触，该检测核酸为单链且不与所述指导RNA的指导序列杂交；以及
(b)测量通过所述工程化的Cas12i核酸酶或工程化的Cas12i效应蛋白切割所述加标签的检测核酸而产生的可检测信号，从而检测所述靶核酸。
32.一种修饰包含靶序列的靶核酸的方法，包括使所述靶核酸与项29或30所述的工程化的CRISPR-Cas12i系统接触；
优选，所述方法在体外进行、离体进行或在体内进行；
进一步优选，所述靶核酸存在于细胞中；
进一步优选，所述细胞是细菌细胞、酵母细胞、哺乳动物细胞、植物细胞或动物细胞；
进一步优选，所述靶核酸是基因组DNA；
进一步优选，所述靶序列与疾病或病症相关；
进一步优选，所述工程化的CRISPR-Cas12i系统包括编码多个crRNA的前体指导RNA阵列，其中每个crRNA包含不同的指导序列。
33.如项29或30所述的工程化的CRISPR-Cas12i系统在制备治疗与个体的细胞中靶核酸相关的疾病或病症的药物中的用途；优选，所述疾病或病症选自下组：癌症、心血管疾病、遗传性疾病、自身免疫疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。
34.一种治疗与个体的细胞中的靶核酸相关的疾病或病症的方法，所述方法包含使用项32所述的方法来修饰所述个体的细胞中的靶核酸，从而治疗所述疾病或病症；优选，所述疾病或病症选自下组：癌症、心血管疾病、遗传性疾病、自身免疫疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。
35.一种修饰包含靶序列的靶核酸的方法，包括使所述靶核酸与项29或30中所述的工程化的CRISPR-Cas12i系统接触。
36.包含如项1-22中任一项所述的工程化的Cas12i核酸酶、如项23-28中任一项所述的工程化的Cas12i效应蛋白的组合物或试剂盒。
37.工程化的细胞，其包含经修饰的靶核酸，其中靶核酸由如项32或35所述的方法修饰。
38.工程化的非人类动物，其包含一种或多种如项37所述的工程化的细胞。
本申请的技术方案取得的有益效果
本申请工程化的Cas12i核酸酶及其效应蛋白具有更高的活性，如切割核酸底物的催化效率及细胞内的基因编辑效率。本申请中的工程化Cas12i核酸酶具有较现有常规Cas基因编辑工具更卓越的哺乳动物细胞(如人类细胞)内的基因编辑效率；例如本申请中的一些示例性Cas12i2核酸酶突变体在人类细胞中多个位点(如62个位点)测试基因编辑效率，发现有57个位点的基因编辑效率超过约60％，平均基因编辑效率接近70％。在一些实施例中，本申请工程化的Cas12i核酸酶及其效应蛋白还具有以下一个或多个优点：蛋白小(1,054aa)，crRNA组分简单，PAM序列简单，并且蛋白自身能加工前体crRNA。另外，本申请提供在高活性工程化Cas12i核酸酶基础上(如SEQ ID NO.8)进一步人工改造的具有更低的脱靶率和更高的特异性的Cas12i核酸酶。这些优点使得本申请高效的工程化的Cas12i核酸酶及其效应蛋白非常适用于在体内进行基因编辑或者基因调控。
附图说明
图1a：将参比Cas12i核酸酶(如SEQ ID NO.1所示的野生型Cas12i核酸酶)中与PAM相互作用的氨基酸替换为带正电的氨基酸，从而提高基因编辑效率。如图所示，E176R，K238R，T447R，E563R四种突变体能够显著地提高在人293T细胞中的基因编辑效率。
图1b：将图1a中能够显著提高基因编辑效率的氨基酸突变(E176R，K238R，T447R，E563R)进行组合，发现组合后的突变体能够展现在人293T细胞中更高的基因编辑效率。
图2：将参比Cas12i核酸酶中参与打开DNA双链的氨基酸替换为带芳香环的氨基酸从而提高基因编辑的效率。如图所示，Q163F、Q163Y、Q163W、N164F、N164Y这些突变体能够显著提高在人293T细胞中的基因编辑效率。
图3a、3b、3c：将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互 作用的氨基酸替换为带正电的氨基酸，从而提高基因编辑的效率。如图3a、3b、3c所示，E323R、L327R、V355R、G359R、G360R、D362R、N391R、Q424R、Q425R、N925R、I926R和G929R等突变体能够显著地提高在人293T细胞中的基因编辑效率。
图3d：将图3a、3b中提高效率的点突变进行组合，发现组合后的突变体能够展现在人293T细胞中更高的基因编辑效率。
图3e：将图3a、3b中提高效率的点突变以及根据分子柔性原理改造突变(L439(L+GG)，I926G)进行组合，发现组合后的突变体能够展现在人293T细胞中更高的基因编辑效率。
图4：将参比Cas12i酶中与DNA-RNA双螺旋相互作用的氨基酸替换为带正电的氨基酸，从而提高基因编辑效率。如图4所示，G116R、E117R、T159R、S161R、E319R、E343R、D958R突变体能够明显地提高在人293T细胞中的基因编辑效率。其中，D958R突变最优。
图5：将图1a～图3e中三种改造策略得到的高效率突变体以及根据分子柔性原理改造突变(L439(L+GG)、I926G)进行组合，发现组合后的突变体能够展现在人293T细胞中更高的基因编辑效率。组合后能够极大地提高基因编辑效率。选出基因编辑效果最佳的突变体并命名为CasXX供后续实验使用。CasXX(SEQ ID NO：8)是具有E176R+K238R+T447R+E563R+N164Y+E323R+D362R突变组合(基于氨基酸序列SEQ ID NO：1的参比Cas12i2)的Cas12i工程酶。
图6a：CasXX在62个人基因组位点的基因编辑效率汇总。PAM＝NTTN。
图6b：CasXX与AsCas12a，BhCas12b v4基因编辑效率的比较。
图6c：CasXX与SpCas9，SaCas9，SaCas9-KKH基因编辑效率的比较。
图6d：CasXX在小鼠Hepa1-6细胞系的基因编辑效率统计。可以看到，CasXX在在小鼠Hepa1-6细胞内65个位点展示出强大的基因编辑能力，平均基因编辑效率超过60％。
图7展示了CasXX在64个人基因位点的基因编辑效率。其中，这64个位点包含的PAM序列覆盖了所有NNNN的组合：NTTN，NTAN，NTCN，NTGN，NATN，NAAN，NACN，NAGN，NCTN，NCAN，NCCN，NCGN，NGTN，NGAN，NGCN，NGGN。如图7所示，CasXX在NTTN，NTAN，NTCN，NTGN，NATN，NAAN，NCTN，NCAN，NGTN这些PAM序列都展现了高效的基因编辑效率，在这些位点的平均基因编辑效率超过40％。
图8展示了野生型Cas12i2蛋白(SEQ ID NO：1)以及CasXX蛋白在体外切割双链DNA的结果。野生型Cas12i2蛋白仅仅对含有NTTN PAM的双链DNA具有部分切割的效率，但是对其余的PAM几乎没有切割活性。但是，CasXX蛋白对含有NTTN，NTAN， NTCN，NTGN，NATN，NAAN，NACN，NCTN，NCAN，NGTN，NGAN PAM的双链DNA都展现了高效的切割效率。几乎能完全切割含有NTTN，NTAN，NTCN，NATN，NAAN，NACN，NCTN，NCAN，NGTN PAM的双链DNA。
图9：Cas12i2(SEQ ID NO：1)与Cas12i1(SEQ ID NO：13)的氨基酸序列的同源性比对。阴影标注的氨基酸代表2种Cas12i蛋白相同的氨基酸，用白色框标注的氨基酸代表2种Cas12i蛋白性质相似的氨基酸。
图10为使用GUIDE-Seq检测CasXX在EMX1-7靶位点的脱靶效应。序列之后的数字代表每种序列被测到的读数(reads)。第一行序列为参考靶序列(SEQ ID NO：77)，下面的序列分别代表靶序列和脱靶序列以及双链DNA标签在靶序列和脱靶序列富集的读数(reads)。该图说明CasXX在EMX1-7位点存在脱靶效应。
图11为在CasXX原有序列的基础上添加新的点突变或者删减原有的点突变，构建新的HF突变体。实验结果表明，R857A，N861A，K807A，N848A，R715A，R719A，K394A，H357A，K844A这些基于CasXX序列的单点突变体能够有效地降低在EMX1-7-OT-1，EMX1-7-OT-2，EMX1-7-OT-3的插入缺失比例。
图12为在CasXX原有序列的基础上添加新的点突变或者删减原有的点突变，构建新的HF突变体。实验结果表明，R857A，N861A，K807A，N848A，R715A，R719A，K394A，H357A，K844A这些基于CasXX序列的单点突变体在使用模拟脱靶的crRNA-RNF2-1-Mis-1/2，crRNA-RNF2-1-Mis-5/6，crRNA-RNF2-1-Mis-17/18和crRNA-RNF2-1-Mis-19/20时能够有效降低插入缺失的比例。
图13为使用荧光报告系统筛选提高基因编辑的特异性的突变体(基于CasXX的HF突变体)的实验流程。将600ng编码Cas蛋白的质粒，300ng编码crRNA的质粒和100ng编码mCherry的质粒，转染到24孔细胞培养皿培养的细胞中。转染3天后，使用流式分析计算每个样品剩余mCherry阳性的比例。计算公式如图展示。
图14为CasXX以及突变体在293T细胞中编辑位于质粒上的mCherry基因。质粒转染后，培养三天，进行流式分析。Cas蛋白的基因编辑效率越高，细胞中mCherry阳性的比例就会越低。实验流程对应图13实验流程示意图；针对mCherry-FM、mCherry-Mis-1/2、mCherry-Mis-5/6以及mCherry-Mis-19/20的间隔(spacer)序列分别如SEQ ID NO：37、38、39和40所示。
图15为在CasXX原有序列的基础上构建新的HF突变体组合。实验结果表明，R857A、R719A、K394A、K844A、R719A/K844A以及R857A/K844A这些基于CasXX序列的突变体能够有效地降低在EMX1-7-OT-1，EMX1-7-OT-2，EMX1-7-OT-3的脱靶插 入缺失比例，并且不牺牲在靶位点的效率。
图16为在CasXX原有序列的基础上构建新的HF突变体组合。实验结果表明，R857A，R719A，K394A，K844A，R719A/K844A以及R857A/K844A这些基于CasXX序列的突变体在使用模拟脱靶的crRNA-RNF2-1-Mis-1/2，crRNA-RNF2-1-Mis-5/6、crRNA-RNF2-1-Mis-17/18和crRNA-RNF2-1-Mis-19/20时能够有效降低插入缺失的比例，并且不牺牲在靶位点的效率。
图17为使用GUIDE-Seq检测CasXX以及CasXX+K394A突变体在CD34-7靶位点的脱靶效应。序列之后的数字代表每种序列被测到的reads数目。第一行序列为参考靶序列(SEQ ID NO：78)，下面的序列分别代表靶序列和脱靶序列以及双链DNA标签在靶序列和脱靶序列富集的reads数目。该图说明CasXX在CD34-7位点存在脱靶效应，但是CasXX+K394A突变体在该位点没有脱靶效应。
具体实施方式
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异来作为区分组件的方式，而是以组件在功能上的差异来作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本发明的较佳实施方式，然所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。除非另有定义，本文中使用的所有技术和科学术语具有与本公开所属领域的普通技术人员通常所理解的相同含义。
I.术语
如本文所用，“效应蛋白”是指具有活性如位点特异性结合活性、单链DNA切割活性、双链DNA切割活性、单链RNA切割活性、DNA或RNA修饰(例如切割，碱基置换、插入、移除)或转录调节活性的蛋白。
如本文所用，“指导RNA”和“gRNA”在本文中可互换使用，是指能够与Cas12i效应蛋白和靶核酸(例如，双链DNA)形成复合物的RNA。本文还考虑了可以被加工成多个crRNA的前体指导RNA阵列。“crRNA”或“CRISPR RNA”包含与靶核酸(例如，双链DNA)的靶序列具有足够互补性的指导序列，其指导CRISPR复合物与靶核酸的靶序列特异性结合。
本文使用的术语“CRISPR阵列”是指包括CRISPR重复和间隔子的核酸(例如DNA)片段，其从第一个CRISPR重复的第一个核苷酸开始并以最后一个(末端)CRISPR重复的最后一个核苷酸结束。典型地，CRISPR阵列中的每个间隔子位于两个重复之间。本文中使用的术语“CRISPR重复”或“CRISPR直接重复”或“直接重复”是指多个短的直接重复序列，其在CRISPR阵列中显示出非常小的序列变化或没有序列变化。适当地，直接重复可以形成茎环结构。
术语“核酸”、“多核苷酸”和“核苷酸序列”可互换使用，是指任何长度的核苷酸的聚合形式，包括脱氧核糖核苷酸、核糖核苷酸、其组合及其类似物。“寡核苷酸”和“低聚核苷酸”可互换使用，是指具有不超过约50个核苷酸的短多核苷酸。如本文所用，“互补性”是指核酸通过传统的沃森-克里克(Watson-Crick)碱基配对与另一核酸形成氢键的能力。互补性百分比表示可与第二种核酸形成氢键(即，沃森-克里克碱基配对)的核酸分子中的残基百分比(例如，10分之5、6、7、8、9、10，分别互补约50％、60％、70％、80％、90％和100％)。“完全互补”是指核酸序列的所有连续残基与第二核酸序列中相同数目的连续残基形成氢键。如本文所用，“基本上互补”是指在约40、50、60、70、80、100、150、200、250个或更多个核苷酸的区域上，互补程度为至少约70％、75％、80％、85％、90％、95％、97％、98％、99％或100中任一个，或指在严格条件下杂交的两种核酸。
如本文所用，用于杂交的“严格条件”是指与靶序列具有互补性的核酸主要与靶序列杂交，而基本上不与非靶序列杂交的条件。严格条件通常是序列依赖性的，并且取决于许多因素而变化。通常，序列越长，序列与其靶序列特异性杂交的温度越高。严格条件的非限制性例子详细描述于Tijssen(1993)，Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes，第一部分第二章″Overview of principles of hybridization and the strategy of nucleic acid probe assay，”Elsevier，N，Y。
“杂交”是指一种或多种多核苷酸反应形成复合物的反应，所述复合物通过核苷酸残基的碱基之间的氢键而稳定化。氢键可通过沃森克里克碱基配对、霍格斯坦(Hoogstein)结合或以任何其他序列特异性方式发生。能够与给定序列杂交的序列称为所述给定序列的“互补体”。
针对核酸序列的“序列同一性百分比(％)”定义为，在通过允许空缺(gaps)来比对序列(如有必要)以实现最大的序列同一性百分比后，候选序列中与特定核酸序列中的核苷酸相同的核苷酸百分比。针对肽、多肽或蛋白质序列的“序列同一性百分比(％)”，是在通过允许空缺来比对序列(如有必要)以实现最大的序列同源性百分比后，候选序列中与特定肽或氨基酸序列中的氨基酸残基相同替换的氨基酸残基的百分比。为了确定氨基酸序列同一 性百分比的目的，比对可以以本领域技术范围内的各种方式来实现，例如，使用诸如BLAST、BLAST-2、ALIGN或MEGALIGN TM(DNASTAR)软件之类的公众可获得的计算机软件。本领域技术人员可以确定用于测量比对的合适参数，包括在所比较序列的全长上实现最大比对所需的任何算法。
术语“多肽”和“肽”在本文可互换使用，是指任何长度的氨基酸的聚合物。所述聚合物可以是直链或支链的，它可以包含经修饰的氨基酸，并且可以被非氨基酸中断。蛋白质可以具有一个或多个多肽。该术语还涵盖已经过修饰的氨基酸聚合物；例如，二硫键的形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作(诸如与标记组分的缀合)。
如本文所用，“变体”解释为分别不同于参比多核苷酸或多肽但保留必要特性的多核苷酸或多肽。多核苷酸的典型变体与另一参比多核苷酸的核酸序列不同。变体核酸序列的变化可以改变或可以不改变参比多核苷酸编码的多肽的氨基酸序列。核苷酸变化可导致参比序列编码的多肽中的氨基酸替换、添加、缺失、融合和截短，如下所述。多肽的典型变体与另一参比多肽在氨基酸序列上不同。通常，差异是有限的，使得参比多肽和变体的序列总体上非常相似，并且在许多区域是相同的。变体和参比多肽的氨基酸序列可以通过一个或多个替换、添加、缺失的任何组合而不同。替换或插入的氨基酸残基可以是或可以不是遗传密码编码的氨基酸残基。多核苷酸或多肽的变体可以是天然存在的(诸如等位基因变体)，或者可以是未知天然存在的变体。多核苷酸和多肽的非天然存在的变体可以通过诱变技术，通过直接合成，以及通过本领域技术人员已知的其他重组方法来制备。
如本文所用，术语“野生型”具有本领域技术人员通常理解的含义，意指当它存在于大自然中时，将其与突变体或变体区分开的、典型形式的生物体、菌株、基因或特征。它可以与自然界中的资源隔离开来，并没有被刻意修饰。
如本文所用，术语“非天然存在”或“工程化的”可互换使用，是指人工参与。当这些术语用于描述核酸分子或多肽时，是指所述核酸分子或多肽至少基本上不含其天然缔合的或天然存在的至少一种其他组分。
如本文所用，术语“直系同源物(orthologue/ortholog)”具有本领域普通技术人员通常理解的含义。作为进一步的指导，本文所指的蛋白质的“直系同源物”是指属于不同物种的蛋白质，其执行与作为其直系同源物的蛋白质相同或相似的功能。
如本文所用，术语“同一性”用于表示两个多肽之间或两个核酸之间的序列匹配。当两个比较序列中的一个位置被相同的碱基或氨基酸单体亚基占据时(例如，两个DNA分子的每个中的一个位置都被腺嘌呤占据，或者两个多肽的每个中的一个位置被赖氨酸占 据)，那么在那个位置每个分子均相同。这两个序列之间的“同一性百分比”是两个序列共有的匹配位置数除以要比较的位置数x 100的函数。例如，如果两个序列的10个位置中有6个匹配，则这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT具有50％的同一性(总共6个位置中有3个匹配)。通常，当两个序列进行比对以产生最大的同一性时，进行这种比较。这种比对可以通过例如Needleman et al.(1970)J.Mol.Biol.48：443-453中的方法来实现，所述方法可方便地通过计算机程序如比对(Align)程序(DNAstar，Inc.)来进行。也可以采用PAM 120权重残基表，使用E.Meyers和W.Miller的算法(Comput.Appl Biosci.，4：11-17(1988))集成到ALIGN程序(2.0版)中。空缺长度罚分12和空缺罚分4，用于确定两个氨基酸序列之间的同一性百分比。此外，可以使用集成到GCG软件包(可从www.gcg.com获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48：444-453(1970))算法，采用Blossum 62矩阵或PAM250矩阵，空缺权重为16、14、12、10、8、6或4，长度权重为1、2、3、4、5或6，以确定两个氨基酸序列之间的同一性百分比。
本文所用的“细胞”应理解为不仅指特定的单个细胞，而且指该细胞的后代或潜在后代。因为由于突变或环境影响，可能在后代中发生某些修饰，所以此类后代可能事实上与亲本细胞不同，但仍包括在本文术语的范围内。
如本文所用，术语“转导”和“转染”包括本领域已知的使用感染剂(如病毒)或其他方式将DNA引入细胞中以表达目的蛋白质或分子的方法。除了病毒或类似病毒的试剂外，还有基于化学的转染方法，如使用磷酸钙、树状聚合物，脂质体或阳离子聚合物(例如DEAE-葡聚糖或聚乙烯亚胺)的转染方法；非化学方法，如电穿孔、细胞挤压(cell squeezing)、声致穿孔(sonoporation)、光学转染、穿刺转染(impalefection)、原生质体融合、质粒递送或转座子；基于颗粒的方法，如使用基因枪、磁转染或磁体辅助转染、颗粒轰击；以及杂交方法(诸如核转染)。
如本文所用，术语“转染的”、“转化的”或“转导的”是指将外源核酸转移或引入宿主细胞的过程。“转染的”、“转化的”或“转导的”细胞是已经用外源核酸转染、转化或转导的细胞。
术语“体内”是指从其中获得细胞的该生物体内。“离体”或“体外”是指从其中获得细胞的该生物体外。
如本文所用，“治疗(treatment/treating)”是用于获得有益的或期望的结果(包括临床结果)的方法。为了本发明的目的，有益的或期望的临床结果包括但不限于以下的一种或多种：减轻由疾病引起的一种或多种症状，减轻疾病的程度，稳定疾病(例如预防或延缓疾 病的恶化)，预防或延缓疾病的扩散(例如转移)，预防或延缓疾病的复发，降低疾病的复发率，延缓或减慢疾病的进展，改善疾病状态，提供疾病的(部分或全部)缓解，减少治疗该疾病所需的一种或多种其他药物的剂量，延缓疾病的进展，提高生活质量，和/或延长生存期。“治疗”还包括减少病症、病况或疾病的病理后果。本发明的方法考虑了这些治疗的方面中的任何一个或多个。
如本文所用，术语“有效量”是指足以治疗特定病症、病况或疾病(如改善、缓解、减轻和/或延迟其一种或多种症状)的化合物或组合物的量。如本领域中所理解的，“有效量”可以以一次或多次给药，即，可能需要单次给药或多此给药来达到期望的治疗终点。
“受试者”、“个体”或“患者”在本文中可互换使用，以达到治疗目的，是指任何归类为哺乳动物的动物，包括人类、家畜和农场动物，以及动物园、农场或宠物动物如狗、马、猫、牛等。在一些实施方案中，所述个体是人类个体。
应理解，本文所述的本发明的实施方案包括“由...组成”和/或“基本上由...组成”的实施方案。在本文中对“约”值或参数的提及包括(并描述了)针对该值或参数本身的变化。例如，提及“大约X”的描述，包括对“X”的描述。
如本文所用，对“不”值或参数的提及通常意指并描述了“除...外”值或参数。例如，所述方法不用于治疗X型癌症，意味着所述方法用于治疗除X型以外的癌症。
如本文所用，术语“大约X-Y”具有与“大约X至大约Y”相同的含义。
如本文和所附权利要求书中所使用的，单数形式“一个/一种(a/an)”和“所述”包括复数对象，除非上下文另外明确指出。还应注意，权利要求可以被撰写为排除任何可选的要素。因此此陈述旨在作为与权利要求要素的叙述结合使用诸如“只”、“仅”等排他性术语的先行基础，或使用“否”的限制。
如本文所用，术语“和/或”在词语诸如“A和/或B”中，旨在既包括A和B；A或B；A(单独)；以及B(单独)。同样地，如本文所用，术语“和/或”在词语诸如“A、B和/或C”中，旨在包括以下每个实施方案：A、B和C；A、B或C；A或C；A或B；B或C；A和C；A和B；B和C；A(单独)；B(单独)；以及C(单独)。
II.Cas12i核酸酶及效应蛋白
工程化的Cas12i核酸酶
在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含以下一种或多种(例如两种、三种、四种或五种)基于参比Cas12i核酸酶的突变，所述突变选自：
(1)将参比Cas12i核酸酶中与PAM相互作用的氨基酸替换为带正电的氨基酸(如R、H或K)；
(2)将参比Cas12i核酸酶中参与打开DNA双链的氨基酸替换为带芳香环的氨基酸(如F、Y或W)；
(3)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的氨基酸替换为带正电的氨基酸(如R、H或K)；
(4)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的氨基酸替换为带正电的氨基酸(如R、H或K)；和
(5)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸(如A、V、I、L、M、F、Y、P、C或W)。在一些实施方式中，所述参比Cas12i核酸酶为天然Cas12i核酸酶，比如氨基酸序列如SEQ ID NO：1所示的野生型Cas12i2核酸酶。在一些实施方式中，所述参比Cas12i核酸酶为Cas12i核酸酶的变体，比如天然变体。在一些实施方式中，所述参比Cas12i核酸酶为不包含上述(1)-(5)中一种或多种突变的工程化的Cas12i(例如Cas12i2或Cas12i1)核酸酶。在一些实施方式中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述氨基酸位置如具有不同于SEQ ID NO：1序列的野生型Cas12i核酸酶的相对应SEQ ID NO：1的氨基酸位置所定义。
如本文所用，“该氨基酸在X位置处，其中所述氨基酸位置如SEQ ID NO：1所示的野生型Cas12i核酸酶的相应氨基酸位置所定义”，或“该氨基酸在X位置处，其中所述氨基酸位置如具有不同于SEQ ID NO：1序列的野生型Cas12i核酸酶的相对应SEQ ID NO：1的氨基酸位置所定义”的含义是：该氨基酸残基位于参比酶Cas12i的某位置处，其相当在SEQ ID NO：1的X位置处，而且参比酶Cas12i的氨基酸序列与SEQ ID NO：1的氨基酸序列基于序列同源性相互对齐。例如，图7示出了CAS12i2(SEQ ID NO：1)与CAS12i1(SEQ ID NO：13)的氨基酸序列的同源性比对。本领域人员可以用本领域常用的软件，如Clustal Omega，将任一参比Cas12i核酸酶的氨基酸序列与SEQ ID NO：1进行序列同一性比较和对齐(alignment)，进而得到与本申请中所述基于SEQ ID NO：1所定义的氨基酸位点相对应的所述参比Cas12i核酸酶中的氨基酸位点。
本申请提供了通过引入氨基酸突变来工程化改造酶的方法，所述氨基酸突变基于上述五种改造原理的任何一种或多种的组合，这导致体外和/或体内酶活性(如DNA单链或双链切割活性)的增加(比如，切割基因效率增加了约100倍)、可识别PAM数目的增加(比如，从图8可以看出，野生型的酶可以识别一种PAM，而基于上述氨基酸突变的 CasXX酶可以识别至少11种PAM)、和/或降低脱靶率及提高特异性(比如，从表18可以看出，HF-Cas相对于CasXX脱靶效率可以降低约99％)。所述工程化Cas12i核酸酶含有一个或多个如以下1)-6)节中所描述的具体突变。在一些实施方式中，本申请中所述的任何一个或多个突变可以与现有的Cas12i突变组合(例如以下7)节中所描述的突变)，以提供具有更高活性的工程化Cas12i核酸酶。
在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸的突变。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸氨基酸替换为带正电的氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸。
在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸的突变；和2)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸的突变；和2)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸；和2)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸。在一些实施方式中，所述工程化的Cas12i核酸酶还包含将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸。在一些实施方式中，所述工程化的Cas12i核酸酶还包含一个或多个柔性区突变(如替换为G，和/或在其后插入一个或两个G)以增加柔性区的柔性。 在一些实施方式中，经过如此修改的Cas12i工程化酶增加了至少约10％的柔性，比如增加了至少约20％、30％、50％、100％、150％、200％、500％、1000％或更高程度的柔性。
在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸的突变；和2)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸氨基酸替换为带正电的氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸；和2)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸氨基酸替换为带正电的氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸；和2)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸氨基酸替换为带正电的氨基酸。在一些实施方式中，所述工程化的Cas12i核酸酶还包含将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸。在一些实施方式中，所述工程化的Cas12i核酸酶还包含一个或多个柔性区突变以增加柔性区的柔性。
在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸的突变；2)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸；和3)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸的突变；2)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸；和3)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸氨基酸替换为带正电的氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸的突变；2)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸；和3)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸氨基酸替换为带正电的氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包 含：1)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸；2)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸；和3)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸氨基酸替换为带正电的氨基酸。在一些实施方式中，所述工程化的Cas12i核酸酶还包含将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸。在一些实施方式中，所述工程化的Cas12i核酸酶还包含一个或多个柔性区突变以增加柔性区的柔性。
在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸的突变；2)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸；3)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸；和4)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸氨基酸替换为带正电的氨基酸。在一些实施方式中，所述工程化的Cas12i核酸酶还包含将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸。在一些实施方式中，提供了一种工程化的Cas12i核酸酶；所述工程化的Cas12i核酸酶包含：1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸的突变；2)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸；3)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸；4)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸氨基酸替换为带正电的氨基酸；和5)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸。在一些实施方式中，所述工程化的Cas12i核酸酶还包含一个或多个柔性区突变以增加柔性区的柔性。
在一些实施方式中，所述工程化的Cas12i核酸酶包含相对于SEQ ID NO：1所示相应氨基酸位置的突变：N164Y+E176R+K238R+E323R+D362R+T447R+E563R(自此命名为“CasXX”)。在一些实施方式中，所述工程化的Cas12i核酸酶包含SEQ ID NO：8的序列。
1)将参比Cas12i核酸酶中与PAM相互作用的氨基酸替换为带正电的氨基酸
在一些实施方式中，所述工程化的Cas12i核酸酶包含一个或多个基于参比Cas12i核酸酶(例如Cas12i2)的突变，所述突变为将参比Cas12i核酸酶中与PAM相互作用的氨基酸替换为带正电的氨基酸(如R、H或K)。在一些实施方式中，所述工程化的Cas12i核酸 酶包含一个、两个、三个、四个、五个、六个、七个、八个或更多所述氨基酸残基的替换。
在一些实施方式中，所述与PAM相互作用的氨基酸是与PAM在三维结构上距离在9埃以内的氨基酸，例如可以为：与PAM在三维结构上距离在9埃以内的氨基酸、与PAM在三维结构上距离在8埃以内的氨基酸、与PAM在三维结构上距离在7埃以内的氨基酸、与PAM在三维结构上距离在6埃以内的氨基酸、与PAM在三维结构上距离在5埃以内的氨基酸、与PAM在三维结构上距离在4埃以内的氨基酸、与PAM在三维结构上距离在3埃以内的氨基酸、与PAM在三维结构上距离在2埃以内的氨基酸、或更近的氨基酸。
PAM与氨基酸的在空间结构上的距离由被解析的Cas蛋白-RNA-DNA三维复合物的3D结构(PDB文件)中的原子之间的距离界定，原子之间的相互距离可以通过PDB文件识别软件显示。在一些实施方式中，所述PAM与氨基酸在空间结构上的距离由氨基酸残基和核苷酸包含的原子之间的最小距离界定。可以用来测量PAM与氨基酸在空间结构上的距离的程序，或PDB文件识别软件在本领域广为人知，包括但不限于PyMOL、ChimeraX、Swiss-pdbviewer等。
在一些实施方式中，所述一个或多个将参比Cas12i核酸酶中与PAM相互作用的氨基酸替换为带正电的氨基酸的突变是在下述位置的一个或多个氨基酸的突变：176、178、226、227、229、237、238、264、447和563。在一些实施方式中，所述一个或多个将参比Cas12i核酸酶中与PAM相互作用的氨基酸替换为带正电的氨基酸的突变是在下述一个或多个氨基酸的突变：E176、E178、Y226、A227、N229、E237、K238、K264、T447和E563。在一些实施方式中，所述一个或多个将参比Cas12i核酸酶中与PAM相互作用的氨基酸替换为带正电的氨基酸的突变是在下述一个或多个氨基酸的突变：E176、K238、T447和E563。在一些实施方式中，所述将参比Cas12i核酸酶中与PAM相互作用的氨基酸替换为带正电的氨基酸的突变位于563号氨基酸残基，例如E563。在一些实施方式中，所述氨基酸位置如SEQ ID NO：1所示的野生型Cas12i核酸酶的相应氨基酸位置所定义。
在一些实施方式中，所述工程化的Cas12i核酸酶包含下述一个或多个氨基酸的突变：E176R、E178R、Y226R、A227R、N229R、E237R、K238R、K264R、T447R和E563R，所述氨基酸位置如SEQ ID NO：1所示的野生型Cas12i核酸酶的相应氨基酸位置所定义。
在本说明书的上下文中，E176的含义是；在所援引的氨基酸序列中(如相对于SEQ ID NO：1)，第176号氨基酸E(谷氨酸)；在此，常见的氨基酸及其三字母和单字母缩写列 举性地说明如下：丙氨酸Ala A；精氨酸Arg R；天冬氨酸Asp D；半胱氨酸Cys C；谷氨酰胺Gln Q；谷氨酸Glu E；组氨酸His H；异亮氨酸Ile I；甘氨酸Gly G；天冬酰胺Asn N；亮氨酸Leu L；赖氨酸Lys K；甲硫氨酸Met M；苯丙氨酸Phe F；脯氨酸Pro P；丝氨酸Ser S；苏氨酸Thr T；色氨酸Trp W；酪氨酸Tyr Y；缬氨酸Val V。
在一些实施方式中，所述将参比Cas12i核酸酶中与PAM相互作用的氨基酸替换为带正电的氨基酸的突变为将参比Cas12i核酸酶中相应的氨基酸残基替换为R、H或K，例如R或K。在一些实施方式中，所述将参比Cas12i核酸酶中与PAM相互作用的氨基酸替换为带正电的氨基酸的突变为将参比Cas12i核酸酶中相应的氨基酸残基替换为R。
在一些实施方式中，所述工程化的Cas12i核酸酶包含以下位点一个或多个氨基酸突变：176R、238R、447R和563R，其中，所述氨基酸位置如SEQ ID NO：1所示的野生型Cas12i核酸酶的相应氨基酸位置所定义。在一些实施方式中，所述工程化的Cas12i核酸酶包含以下一个或多个基于参比Cas12i核酸酶的突变：E176、K238、T447和E563；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化的Cas12i核酸酶包含以下一个或多个基于参比Cas12i核酸酶的突变：E176R、K238R、T447R和E563R；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化的Cas12i核酸酶包含E563R突变；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，为改善基因编辑效率的目的，也可以使用与上述经工程化的Cas12i核酸酶(将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸)具有至少约85％(例如任一至少约86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的工程化的Cas12i核酸酶。
在本说明书的上下文中，E176R表示的含义是，在所援引的氨基酸序列中，将第176号氨基酸E(谷氨酸)替换为R(精氨酸)。
在一些实施方式中，所述工程化Cas12i核酸酶包含任一个在下述氨基酸残基位置的突变或突变组合：(i)176、238、264、447、563、176+238、176+447、176+563、238+447、238+563、447+563、176+238+447、176+238+563、176+447+563、238+447+563、176+238+447+563；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸包括替换为R、H或K，例如R或K，优选的，例如R。在一些实施方式中，所述工程化Cas12i核酸酶包含任一个在下述氨基酸残基的突变或突变组合：在一些实施方式中，所述工程化Cas12i核酸酶包含任一个在下述氨基酸残基的突 变或突变组合：E176、K238、E264、T447、E563、E176+K238、E176+T447、E176+E563、K238+T447、K238+E563、T447+E563、E176+K238+T447、E176+K238+E563、E176+T447+E563、K238+T447+E563、和E176+K238+T447+E563；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方式中，所述工程化Cas12i核酸酶包含下述突变/突变组合的任一个：E176R、K238R、E264R、T447R、E563R、E176R+K238R、E176R+T447R、E176R+E563R、K238R+T447R、K238R+E563R、T447R+E563R、E176R+K238R+T447R、E176R+K238R+E563R、E176R+T447R+E563R、K238R+T447R+E563R、和E176R+K238R+T447R+E563R；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含下述突变/突变组合的任一个：E563R、E176R+T447R、E176R+E563R、K238R+E563R、E176R+K238R+T447R、E176R+K238R+E563R、E176R+T447R+E563R、和E176R+K238R+T447R+E563R；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R+K238R+T447R+E563R突变组合；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。为改善基因编辑效率的目的，也可以使用与上述经工程化的Cas12i核酸酶(将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸)具有至少约85％(例如任一至少约86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的工程化的Cas12i核酸酶。
2)将参比Cas12i核酸酶中参与打开DNA双链的氨基酸替换为带芳香环的氨基酸
在一些实施方式中，所述工程化的Cas12i核酸酶包含一个或多个基于参比Cas12i核酸酶(例如Cas12i2)的突变，所述突变为将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸(如F、Y或W)。在一些实施方式中，所述工程化的Cas12i核酸酶包含一个、两个、三个、四个、五个、六个、或更多所述氨基酸残基的替换。
其中，所述参与打开DNA双链的一个或多个氨基酸是与PAM中相对于靶标链的3’端最后一个碱基对相互作用的氨基酸。例如，Cas12i2识别的PAM序列是5’-NTTN-3’碱基对，其中PAM序列中3’末端的N碱基与靶标链所形成的碱基对就是文本所述的“与PAM中相对于靶标链的3’端最后一个碱基对，”而该碱基对之后就是靶向位点的序列。
在一些实施方式中，所述一个或多个参与打开DNA双链的氨基酸位于下述位置： 163和/或164；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述一个或多个参与打开DNA双链的氨基酸是下述一个或多个氨基酸：Q163、N164；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述参与打开DNA双链的氨基酸为N164；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方式中，所述参与打开DNA双链的氨基酸被替换为F、Y或W。在一些实施方式中，所述参与打开DNA双链的氨基酸被替换为F。在一些实施方式中，所述参与打开DNA双链的氨基酸被替换为Y。
在一些实施方式中，所述工程化Cas12i核酸酶包含下述任一个或多个氨基酸残基的突变：163F、163Y、163W、164W、164F或164Y；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含下述任一个或多个氨基酸残基的突变：163F、163Y、163W、164F或164Y；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方式中，所述工程化Cas12i核酸酶包含下述任一个突变：Q163和/或N164；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含下述任一个突变：Q163F、Q163Y、Q163W、N164W、N164F或N164Y；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含下述任一个突变：Q163F、Q163Y、Q163W、N164F或N164Y；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含N164Y或N164F突变；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含N164Y突变；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施例中，为实现改善基因编辑效率的目的，也可以使用与上述工程化的Cas12i核酸酶(将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸)具有至少约85％(例如任一至少约86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的工程化的Cas12i核酸酶。
3)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的氨基酸替换为 带正电的氨基酸
在一些实施方式中，所述工程化的Cas12i核酸酶包含一个或多个基于参比Cas12i核 酸酶(例如Cas12i2)的突变，所述突变为将参比Cas12i酶中位于RuvC结构域并与单链DNA底物相互作用的氨基酸替换为带正电的氨基酸(如R、H或K)。在一些实施方式中，所述工程化的Cas12i酶包含一个、两个、三个、四个、五个、六个或更多所述氨基酸残基的替换。
其中，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是在三维结构上与单链DNA底物距离在9埃以内的氨基酸，例如可以为：在三维结构上与单链DNA底物距离在8埃以内的氨基酸、在三维结构上与单链DNA底物距离在7埃以内的氨基酸、在三维结构上与单链DNA底物距离在6埃以内的氨基酸、在三维结构上与单链DNA底物距离在5埃以内的氨基酸、在三维结构上与单链DNA底物距离在4埃以内的氨基酸、在三维结构上与单链DNA底物距离在3埃以内的氨基酸、在三维结构上与单链DNA底物距离在2埃以内的氨基酸、或更近的氨基酸。
RuvC结构域是Cas12i蛋白中负责切割单链DNA或者双链DNA的酶活结构域。在蛋白质的一级序列中，Cas12i的RuvC结构域被分为3个部分：RuvC-1，RuvC-2以及RuvC-3。这3个部分在三维结构中相邻近，一同组成具有酶切活性的催化口袋。Cas12i2的三维晶体结构，其结构域组成，及与DNA底物相互作用的描述见Huang X.et al.，Nature Communications，11，Article number：5241(2020)。Cas12i1的三维晶体结构，其结构域组成，及与DNA底物相互作用的描述见Zhang H.et al.Nature Structural&Molecular Biology 27，1069-1076(2020)。可以通过同源结构比较和建模(homology modeling)通过已知的Cas12i三维晶体结构，得到参比Cas12i和底物相互作用的三维结构模型。实施例3中描述了一种建模方式以得到Cas12i2中位于RuvC结构域并与单链DNA底物距离在9埃以内的氨基酸。
在一些实施方式中，RuvC结构域中的氨基酸与单链DNA底物在空间结构上的距离可以由被解析的Cas蛋白-RNA-DNA三维复合物的3D结构(PDB文件)中的原子之间的距离界定，原子之间的相互距离可以通过PDB文件识别软件显示。在一些实施方式中，所述RuvC结构域中的氨基酸与单链DNA底物在空间结构上的距离由氨基酸残基和核苷酸包含的原子之间的最小距离界定。可以用来测量RuvC结构域中的氨基酸与单链DNA底物在空间结构上的距离的程序，或PDB文件识别软件在本领域广为人知，包括但不限于PyMOL、ChimeraX、Swiss-pdbviewer等。
在一些实施方式中，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是下述位置的一个或多个氨基酸：323、327、355、359、360、361、362、388、390、391、392、393、414、417、418、421、424、425、650、652、653、696、705、 708、709、751、752、755、840、848、851、856、885、897、925、926、928、929、932、和1022。在一些实施方式中，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是下述一个或多个氨基酸：E323、L327、V355、G359、G360、K361、D362、L388、N390、N391、F392、K393、Q414、L417、L418、K421、Q424、Q425、S650、E652、G653、I696、K705、K708、E709、L751、S752、E755、N840、N848、S851、A856、Q885、M897、N925、I926、T928、G929、Y932、A1022。在一些实施方式中，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是下述一个或多个氨基酸：E323、D362、L388、N391、L417、Q424、Q425、N925、I926、和G929。在一些实施方式中，所述氨基酸位置如SEQ ID NO：1所示的野生型Cas12i核酸酶的相应氨基酸位置所定义。
在一些实施方式中，所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为R、H或K(例如R或K)的突变。在一些实施方式中，所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为R的突变。
在一些实施方式中，所述工程化的Cas12i核酸酶包含下述一个或多个氨基酸突变或突变组合：N390R、N391R、F392R、L751R、E755R、N840R、N848R、S851R、A856R、Q885R、M897R、I926R、G929R、Y932R、E323R、L327R、V355R、G359R、G360R、K361R、D362R、Q414R、K421R、Q425R、S650R、E652R、K705R、K708R、E709R、S752R、N925R、T928R、E323R+D362R、E323R+Q425R、E323R+I926R、Q425R+I926R、E323R+D362R+Q425R、E323R+D362R+I926R、E323R+Q425R+I926R、E323R+D362R+Q425R+I926R、D362R+I926R、N925R+I926R、D362R+N925R+I926R、D362R+N925R；所述氨基酸位置如SEQ ID NO：1所示的野生型Cas12i核酸酶的相应氨基酸位置所定义。
在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述氨基酸残基位置的突变或突变组合：E323、D362、Q425、N925、I926和G929；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述氨基酸残基位置的突变或突变组合：E323、D362、Q425、N925、I926、E323+D362、E323+Q425、E323+I926、D362+Q425、D362+N925、D362+I926、Q425+I926、N925+I926、E323+D362+Q425、E323+D362+I926、E323+Q425+I926、D362+N925+I926、D362+Q425+I926、E323+D362+Q425+I926；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述突变为将所述 位置氨基酸残基替换为R、H或K(如R)的突变。在一些实施方式中，所述工程化Cas12i核酸酶包含下述任何一个氨基酸或氨基酸组合：323R、362R、425R、925R、926R、323R+362R、323R+425R、323R+926R、362R+425R、362R+926R、425R+926R、925R+926R、323R+362R+425R、323R+362R+926R、323R+425R+926R、362R+925R+926R、323R+362R+425R+926R、和362R+425R+926R；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述突变或突变组合：E323R、D362R、Q424R、Q425R、N925R、I926R、和G929R；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述突变或突变组合：E323R、D362R、Q425R、N925R、I926R、E323R+D362R、E323R+Q425R、E323R+I926R、D362R+Q425R、Q425R+I926R、D362R+I926R、N925R+I926R、E323R+D362R+Q425R、E323R+D362R+I926R、E323R+Q425R+I926R、D362R+N925R+I926R、D362R+Q425R+I926R、和E323R+D362R+Q425R+I926R；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含I926R突变；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含E323R+D362R突变；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施例中，为实现改善基因编辑效率的目的，也可以使用与上述工程化的Cas12i核酸酶(将参比Cas12i核酸酶位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸)具有至少约85％(例如任一至少约86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的工程化的Cas12i核酸酶。
4)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为带正 电的氨基酸
在一些实施方式中，所述工程化的Cas12i核酸酶包含一个或多个基于参比Cas12i核酸酶(例如Cas12i2)的突变，所述突变为将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为带正电的氨基酸(如R、H或K)。在一些实施方式中，所述工程化的Cas12i酶包含一个、两个、三个、四个、五个、六个、或更多所述氨基酸残基的替换。
其中，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是在三维结构上与 DNA-RNA双螺旋距离在9埃以内的氨基酸，例如可以为：在三维结构上与DNA-RNA双螺旋距离在8埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在7埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在6埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在5埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在4埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在3埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在2埃以内的氨基酸、或更近的氨基酸。某些Cas核酸酶的工作原理如下：Cas与指导RNA(如crRNA)形成复合体，其中crRNA和靶向DNA相互配对形成的DNA-RNA双螺旋，并与Cas核酸酶相互作用，打开双链靶向DNA，并形成R-loop，得以使Cas的酶切活性位点完成对dsDNA的切割。Cas12i2的三维晶体结构，其结构域组成，及与DNA-RNA双螺旋相互作用的描述见Huang X.et al.，Nature Communications，11，Article number：5241(2020)。
在一些实施方式中，DNA-RNA双螺旋和Cas氨基酸在空间结构上的距离可以由被解析的Cas蛋白-RNA-DNA三维复合物的3D结构(PDB文件)中的氨基酸残基和核苷酸包含的原子之间的最小距离界定，原子之间的相互距离可以通过PDB文件识别软件显示。在一些实施方式中，所述Cas氨基酸与DNA-RNA双螺旋在空间结构上的距离由原子假设位置的距离界定。可以用来测量Cas氨基酸与DNA-RNA双螺旋在空间结构上的距离的程序，或PDB文件识别软件在本领域广为人知，包括但不限于PyMOL、ChimeraX、Swiss-pdbviewer等。
在一些实施方式中，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是下述位置的一个或多个氨基酸：116、117、156、159、160、161、247、293、294、297、301、305、306、308、312、313、316、319、320、343、348、349、427、433、438、441、442、679、683、691、782、783、797、800、852、853、855、861、865、957、958。在一些实施方式中，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是下述一个或多个氨基酸：G116、E117、A156、T159、E160、S161、E247、G293、E294、N297、T301、I305、K306、T308、N312、F313、Q316、E319、Q320、E343、E348、E349、D427、K433、V438、N441、Q442、N679、E683、E691、D782、E783、E797、E800、M852、D853、L855、N861、Q865、S957、D958。在一些实施方式中，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是下述一个或多个氨基酸：G116、E117、T159、S161、E319、E343、或D958。在一些实施方式中，所述与DNA-RNA双螺旋相互作用的氨基酸是D958。在一些实施方式中，所述氨基酸位置如SEQ ID NO：1所示的野生型Cas12i核酸酶的相应氨基酸位置所定义。
在一些实施方式中，所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为R、H或K(例如R或K)的突变。在一些实施方式中，所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中参与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为为R的突变。
在一些实施方式中，所述工程化的Cas12i核酸酶包含下述一个或多个氨基酸突变：G116R、E117R、A156R、T159R、S161R、T301R、I305R、K306R、T308R、N312R、F313R、D427R、K433R、V438R、N441R、Q442R、M852R、L855R、N861R、Q865R、E160R、Q316R、E319R、Q320R、E247R、E343R、E348R、E349R、N679R、E683R、E691R、D782R、E783R、E797R、E800R、D853R、S957R、D958R、G293R、E294R、N297R；所述氨基酸位置如SEQ ID NO：1所示的野生型Cas12i核酸酶的相应氨基酸位置所定义。
在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述氨基酸残基位置的突变或突变组合：G116、E117、T159、S161、E319、E343、或D958；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述突变为将所述位置氨基酸残基替换为R、H或K(如R)的突变。在一些实施方式中，所述工程化Cas12i核酸酶包含下述任何一个位点氨基酸或氨基酸突变组合：116R、117R、159R、161R、319R、343R、或958R；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述突变或突变组合：G116R、E117R、T159R、S161R、E319R、E343R、或D958R；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含D958R突变；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施例中，为实现改善基因编辑效率的目的，也可以使用与上述工程化的Cas12i核酸酶具有至少约85％(例如任一至少约86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的工程化的Cas12i核酸酶。
5)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的 氨基酸替换为疏水氨基酸
在一些实施方式中，所述工程化的Cas12i核酸酶包含一个或多个基于参比Cas12i核酸酶(例如Cas12i2)的突变，所述突变为将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸(如A、V、I、L、M、F、 Y、P、C或W)。所述突变(在此也称为“高特异性”或“HF”突变)可以降低Cas12i核酸酶的脱靶率(即提高特异性)。在一些实施方式中，CasXX-HF相对于CasXX脱靶率可以降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、99％或降低更多。在一些实施方式中，所述工程化的Cas12i酶包含一个、两个、三个、四个、五个、六个或更多所述氨基酸残基的替换。
其中，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是在三维结构上与DNA-RNA双螺旋距离在9埃以内的氨基酸，例如可以为：在三维结构上与DNA-RNA双螺旋距离在8埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在7埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在6埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在5埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在4埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在3埃以内的氨基酸、在三维结构上与DNA-RNA双螺旋距离在2埃以内的氨基酸、或更近的氨基酸。Cas12i2的三维晶体结构，其结构域组成，及与DNA-RNA双螺旋相互作用的描述见Huang X.et al.，Nature Communications，11，Article number：5241(2020)。
在一些实施方式中，所述与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸是下述位置的一个或多个氨基酸：119、164、297、308、309、312、346、357、394、395、402、441、433、565、715、719、766、782、807、841、844、845、848、857、861和865。在一些实施方式中，所述与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸是下述一个或多个氨基酸：Y119、Y164、N297、T308、R309、N312、S346、H357、K394、E395、R402、N441、K433、S565、R715、R719、S766、D782、K807、N841、K844、K845、N848、R857、N861和Q865。在一些实施方式中，所述与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸是下述一个或多个氨基酸：R857、R861、K807、N848、R715、R719、K394、H357和K844。在一些实施方式中，所述与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸是下述一个或多个氨基酸：R857、R719、K394、和K844。其中，如上所述氨基酸位置如SEQ ID NO：1或8所示的相应氨基酸位置所定义。
在一些实施方式中，所述工程化的Cas12i核酸酶包含下述一个或多个突变：S565A、N297A、Q865A、T308A、R309A、N312A、N441A、R857A、N861A、Y119F、K433A、K807A、N841A、N848A、K845A、D782A、R715A、R719A、S766A、K394A、H357A、K844A、E395A、S346A、R402A、Y164N；其中，氨基酸位置编号如SEQ ID NO：1或8所示的相应氨基酸位置所定义。
在一些实施方式中，所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中位于357、394、715、719、807、844、848、857、861的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸的突变。在一些实施方式中，所述疏水氨基酸选自A、V、L、I、P和F，例如A、V、L或I。在一些实施方式中，所述工程化的Cas12i核酸酶包含将参比Cas12i核酸酶中位于H357、K394、R715、R719、K807、K844、N848、R857和/或R861的一个或多个极性或带正电荷的氨基酸替换为A的突变。其中，所述氨基酸位置编号如SEQ ID NO：1或8所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含下述任何一个位置氨基酸或氨基酸组合的突变：H357A、K394A、R715A、R719A、K807A、K844A、N848A、R857A、和R861A；其中，氨基酸位置编号如SEQ ID NO：1或8所示的相应氨基酸位置所定义。
在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述氨基酸残基位置的突变或突变组合：857、719、394、或844；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述突变为将所述位置氨基酸残基替换为疏水氨基酸(如A)的突变。在一些实施方式中，所述工程化Cas12i核酸酶包含下述任何一个位置氨基酸或氨基酸组合的突变：R857、R719、K394、或K844；其中，氨基酸位置编号如SEQ ID NO：1或8所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述氨基酸残基位置的突变或突变组合：R857、R719、K394、K844、R719+K394、K394+K844、R857+K394、R719+K844、R857+R719、R857+K844、R719+K394+K844、R857+R719+K394、R857+K394+K844、R857+R719+K844、R857+R719+K394+K844；其中，氨基酸位置编号如SEQ ID NO：1或8所示的相应氨基酸位置所定义；所述突变为将所述位置氨基酸残基替换为疏水氨基酸(如A)的突变。在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述氨基酸残基位置的突变或突变组合：R857A、R719A、K394A、K844A、R719A+K394A、K394A+K844A、R857A+K394A、R719A+K844A、R857A+R719A、R857A+K844A、R719A+K394A+K844A、R857A+R719A+K394A、R857A+K394A+K844A、R857A+R719A+K844A、R857A+R719A+K394A+K844A；其中，氨基酸位置编号如SEQ ID NO：1或8所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述突变或突变组合：R857A、R719A、K394A、或K844A；其中，氨基酸位置编号如SEQ ID NO：1或8所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含K844A突变；其中，氨基酸位置编号如SEQ ID NO：1或8所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含 R719A和K844A突变；其中，氨基酸位置编号如SEQ ID NO：1或8所示的相应氨基酸位置所定义。在一些实施方式中，所述工程化Cas12i核酸酶包含R857A和K844A突变；其中，氨基酸位置编号如SEQ ID NO：1或8所示的相应氨基酸位置所定义。在一些实施例中，为实现改善基因编辑效率的目的，也可以使用与上述工程化的Cas12i核酸酶(将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸)具有至少约85％(例如任一至少约86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的工程化的Cas12i核酸酶。
6)其它突变
如未特殊说明，本文所述突变可以包括一个或多个：插入、删除、置换，可以是单个氨基酸或多个氨基酸的突变。
在1)至5)节中描述的任一个或者多个突变可以结合任一个或多个增加Cas12i活性的已知突变，例如靶标结合、双链切割活性、切口酶活性和/或基因编辑活性。示例行的突变例如可见于下述文献PCT/CN2020/0134249和CN 112195164 A，这些文献通过整体引用而并入本文。在1)至5)节中描述的任一个或者多个突变也可以结合任一个或多个降低Cas12i活性的已知突变，例如靶标结合、双链切割活性、切口酶活性和/或基因编辑活性。
在一些实施方式中，所述工程化的Cas12i核酸酶(如包含上述1)至5)节中一个或者多个突变的工程化的Cas12i核酸酶)还包含一个或多个柔性区突变，所述突变增加了(比如增加至少约任一10％、20％、50％、60％、70％、80％、90％、1倍、1.1倍、1.2倍、1.5倍、2倍、3倍、4倍、5倍、10倍、20倍、50倍、100倍或更多)参比Cas12i核酸酶(或所述包含(1)-(5)节中任一个或者多个突变的工程化的Cas12i核酸酶)中的柔性区的柔性(flexibility)。所述参比Cas12i核酸酶中的柔性区可以使用本领域中已知的任何方法来确定。在一些实施方案中，仅基于所述参比Cas12i核酸酶的氨基酸序列确定多个柔性区。在一些实施方案中，基于所述参比Cas12i核酸酶的结构信息确定多个柔性区，包括例如二级结构、晶体结构、NMR结构等。
此处所述的工程化改造Cas12i核酸酶柔性区的方法包括：(a)获得多种工程化的Cas12i核酸酶，每种工程化的Cas12i核酸酶包含一个或多个突变，所述突变增加了参比Cas12i核酸酶的一个或多个柔性区中的柔性区柔性；以及(b)从所述多种工程化的Cas12i核酸酶中选择一种或多种工程化的Cas12i核酸酶，其中所述一种或多种工程化的Cas12i核酸酶与所述参比Cas12i核酸酶相比具有增加的活性(例如靶标结合、双链切割活性、切 口酶活性和/或基因编辑活性)。在一些实施方案中，所述方法还包括确定所述参比Cas12i核酸酶中的一个或多个柔性区。在一些实施方案中，所述方法还包括在真核细胞诸如哺乳动物细胞(例如人细胞)中测量所述工程化的Cas12i核酸酶的活性。
在一些实施方案中，使用选自下组的程序确定多个柔性区：PredyFlexy、FoldUnfold、PROFbval、Flexserv、FlexPred、DynaMine和Disomine。在一些实施方案中，所述一个或多个柔性区位于无规卷曲处。在一些实施方案中，所述一个或多个柔性区在参比Cas12i核酸酶(或所述包含(1)-(5)节中任一个或者多个突变的工程化的Cas12i核酸酶)的与DNA和/或RNA相互作用的结构域中。在一些实施方案中，所述柔性区的长度为至少约5个(例如5个)氨基酸。
在一些实施方案中，所述一个或多个突变包括在柔性区中插入一个或多个(例如2个)甘氨酸(G)残基。在一些实施方案中，所述一个或多个G残基插入在柔性区中的柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、丝氨酸(S)、天冬酰胺(N)、天冬氨酸(D)、组氨酸(H)、蛋氨酸(M)、苏氨酸(T)、谷氨酸(E)、谷氨酰胺(Q)、赖氨酸(K)、精氨酸(R)、丙氨酸(A)和脯氨酸(P)。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G＞S＞N＞D＞H＞M＞T＞E＞Q＞K＞R＞A＞P。在一些实施方案中，所述一个或多个突变包括用一个或多个G残基替换一个或多个非G残基。
在一些实施方案中，所述一个或多个突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：亮氨酸(L)、异亮氨酸(I)、缬氨酸(V)、半胱氨酸(C)、酪氨酸(Y)、苯丙氨酸(F)和色氨酸(W)。
在一些实施方案中，所述活性是位点特异性核酸酶活性。在一些实施方案中，所述活性是真核细胞(例如人细胞)中的基因编辑活性。在一些实施方案中，所述基因编辑效率是使用如下方法来测量的：T7核酸内切酶1(T7E1)测定、靶DNA的测序、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。
在一些实施方案中，所述工程化Cas12i核酸酶(例如所述包含(1)-(5)节中任一个或者多个突变的工程化的Cas12i核酸酶)包含一个或多个柔性区突变，所述柔性区突变增加了参比Cas12i核酸酶(如Cas12i2核酸酶、或所述包含(1)-(5)节中任一个或者多个突变的工程化的Cas12i核酸酶)中柔性区的柔性，所述柔性区选自对应于以下的区的组：氨基酸残基228-232、氨基酸残基439-443、氨基酸残基478-482、氨基酸残基500-504、氨基酸残基775-779和氨基酸残基925-929，其中所述氨基酸残基编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方案中，所述柔性区选自氨基酸残基439-443或氨基酸残基925-929，其中所述氨基酸残基编号如SEQ ID NO：1所示的相应氨基酸位置所定 义。在一些实施方案中，所述参比Cas12i酶是Cas12i2(SEQ ID NO：1)。在一些实施方案中，所述一个或多个柔性区突变包括在所述柔性区中插入一个或多个(例如2个)G残基。在一些实施方案中，所述一个或多个G残基插入在所述柔性区中柔性氨基酸残基的N末端，其中所述柔性氨基酸残基选自下组：G、S、N、D、H、M、T、E、Q、K、R、A和P。在一些实施方案中，根据以下优先级选择所述柔性氨基酸残基：G＞S＞N＞D＞H＞M＞T＞E＞Q＞K＞R＞A＞P。在一些实施方案中，所述一个或多个柔性区突变包括用G残基替换柔性区中的疏水氨基酸残基，其中所述疏水氨基酸残基选自下组：A、V、I、L、M、F、Y、P、C或W；优选的，选自：L、I、V、C、Y、F和W。
在一些实施方式中，所述柔性区突变位于439和/或926。在一些实施方式中，它们是下述一个或多个氨基酸：L439、I926。其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方案中，所述工程化的Cas12i核酸酶(例如所述包含(1)-(5)节中任一个或者多个突变的工程化的Cas12i核酸酶)包含926G和/或439(L+G)突变。在一些实施方案中，所述工程化的Cas12i核酸酶包含一个或多个下列柔性区突变：I926G、L439(L+G)和L439(L+GG)。在一些实施方案中，所述工程化的Cas12i核酸酶包含I926G突变。在一些实施方案中，所述工程化的Cas12i核酸酶包含L439(L+G)突变。在一些实施方案中，所述工程化的Cas12i核酸酶包含L439(L+GG)突变。其中，所述氨基酸残基编号基于SEQ ID NO：1。
在本说明书的上下文中，L439(L+G)的含义是，在所援引的氨基酸序列中(如SEQ ID NO：1)，在第439号氨基酸后插入一个甘氨酸(G)，原有第439位的L序列不变；在本申请的上下文以及附图中，有时也表示为439G。而L439(L+GG)的含义是，在所援引的氨基酸序列中，在第439号氨基酸后插入两个甘氨酸(GG)，原有第439位的L序列不变；在本申请的上下文中以及附图中，有时也表示为439GG。
在一些实施方式中，所述工程化Cas12i核酸酶(例如所述包含(1)-(5)节中任一个或者多个突变的工程化的Cas12i核酸酶)包含任一个下述氨基酸残基位置的突变或突变组合：926、439、925+926、362+925+926、439+926、323+362+926(例如I926、L439、N925+I926、D362+N925+I926、L439+I926、E323+D362+I926)；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述位于氨基酸位置323、362、925或926的突变为将所述位置氨基酸残基替换为R、H或K(如R)的突变；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述位于氨基酸位置439或926的突变为将所述位置氨基酸残基替换为G或在所述 氨基酸残基后插入G或GG的突变；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方式中，所述工程化Cas12i核酸酶(例如所述包含(1)-(5)节中任一个或者多个突变的工程化的Cas12i核酸酶)包含任一个下述氨基酸残基或氨基酸残基组合的突变：926G、439(L+GG)、925R+926G、362R+925R+926G、439(L+GG)+926R、或323R+362R+926G；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述突变或突变组合：I926G、L439(L+GG)、L439(L+GG)+I926R、N925R+I926G、D362R+N925R+I926G、E323R+D362R+I926G；其中氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方案中，为实现改善基因编辑效率的目的，也可以使用与上述工程化的Cas12i核酸酶(例如所述包含(1)-(5)节中任一个或者多个突变的工程化的Cas12i核酸酶，和/或包含上述柔性区突变的工程化的Cas12i核酸酶)具有至少约85％(例如任一至少约86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的工程化的Cas12i核酸酶。
7)组合突变
利用根据本说明书中1)-6)节所描述的突变以及表1至表5，表9、表12、表14、表16-18中的一个或多个氨基酸替换/插入的组合而得到的工程化的Cas12i核酸酶都在本申请的请求保护范围之下。
在一些实施方案中，所述工程化的Cas12i核酸酶包含任一个下述氨基酸残基位置的突变或突变组合：164、176、238、323、357、362、394、439、447、563、715、719、807、844、848、857、861、925、926、958、176+238+447+563、323+362、176+238+447+563+164、176+238+447+563+926、176+238+447+563+323+362、164+926、164+323+362、176+238+447+563+164+926、176+238+447+563+164+323+362、176+238+447+563+164+926+323+362、176+238+447+563+164+323+362+926+439、719+844、857+844、362+926、925+926、362+925+926、439+926、323+362+926；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。在一些实施方式中，所述位于氨基酸位置176、238、323、362、447、563、926、或958的突变为将所述位置氨基酸残基替换为R、H或K(如R)的突变。在一些实施方式中，所述位于氨基酸位置164 的突变为将所述位置氨基酸残基替换为Y或F(如Y)的突变。在一些实施方式中，所述位于氨基酸位置439或926的突变为将所述位置氨基酸残基替换为G或在所述氨基酸残基后插入G或GG的突变。在一些实施方式中，所述位于氨基酸位置357、394、715、719、807、844、848、857、或861的突变为将所述位置氨基酸残基替换为疏水氨基酸(如A)。其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述氨基酸残基或氨基酸残基组合的突变：N164、E176；K238；E323、D362、T447；E563、I926、I926、D958、L439、R857、N861、K807、N848、R715、R719、K394、H357、K844、E176+K238+T447+E563、E323+D362、E176+K238+T447+E563+N164、E176+K238+T447+E563+I926、E176+K238+T447+E563+E323+D362、N164+I926、N164+E323+D362、E176+K238+T447+E563+N164+I926、E176+K238+T447+E563+N164+E323+D362、E176+K238+T447+E563+N164+I926+E323+D362、E176+K238+T447+E563+N164+E323+D362+I926、E176+K238+T447+E563+N164+E323+D362+I926+L439、E176+K238+T447+E563+N164+E323+D362+I926+L439；
E176+K238+T447+E563+N164+D958；
E176+K238+T447+E563+I926+D958；
E176+K238+T447+E563+E323+D362+D958；
N164+I926+D958；
N164+E323+D362+D958；
E176+K238+T447+E563+N164+D958；
E176+K238+T447+E563+N164+I926+D958；
E176+K238+T447+E563+N164+E323+D362+D958；
E176+K238+T447+E563+N164+I926+E323+D362+D958；
E176+K238+T447+E563+N 164+E323+D362+I926+D958；
E176+K238+T447+E563+N164+E323+D362+I926+L439+D958；
E176+K238+T447+E563+N164+E323+D362+I926+L439+D958；
R719+K844；
R857+K844；
E176+K238+T447+E563+N164+E323+D362+R857；
E176+K238+T447+E563+N164+E323+D362+N861；
E176+K238+T447+E563+N164+E323+D362+K807；
E176+K238+T447+E563+N164+E323+D362+N848；
E176+K238+T447+E563+N164+E323+D362+R715；
E176+K238+T447+E563+N164+E323+D362+R719；
E176+K238+T447+E563+N164+E323+D362+K394；
E176+K238+T447+E563+N164+E323+D362+H357；
E176+K238+T447+E563+N164+E323+D362+K844；
E176+K238+T447+E563+N164+E323+D362+R719+K844；
E176+K238+T447+E563+N164+E323+D362+R857+K844；或
E176+K238+T447+E563+N164+E323+D362+K394；
其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述突变或突变组合：
N164Y；E176R；K238R；E323R；D362R；T447R；E563R；I926R；D958R；L439(L+G)；
L439(L+GG)；R857A；N861A；K807A；N848A；R715A；R719A；K394A；H357A；K844A；
R719A+K844A；N164Y+I926R；E323R+D362R；R857A+K844A；R857A+K844A；
N164Y+I926R+D958R；N164Y+E323R+D362R；N164Y+E323R+D362R+D958R；
E176R+K238R+T447R+E563R；E176R+K238R+T447R+E563R+N164Y；
E176R+K238R+T447R+E563R+I926R；E176R+K238R+T447R+E563R+E323R+D362R；
E176R+K238R+T447R+E563R+N164Y+I926R；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R；
E176R+K238R+T447R+E563R+N164Y+I926R+E323R+D362R；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+L439(L+GG)；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+L439(L+G)；
E176R+K238R+T447R+E563R+N164Y+D958R；
E176R+K238R+T447R+E563R+I926R+D958R；
E176R+K238R+T447R+E563R+E323R+D362R+D958R；
E176R+K238R+T447R+E563R+N164Y+D958R；
E176R+K238R+T447R+E563R+N164Y+I926R+D958R；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+D958R；
E176R+K238R+T447R+E563R+N164Y+I926R+E323R+D362R+D958R；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+D958R；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+L439(L+GG)+D958R；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+L439(L+G)+D958R；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+R857A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+N861A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+K807A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+N848A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+R715A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+R719A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+K394A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+H357A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+K844A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+R719A+K844A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+R857A+K844A；或
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+K394A；
其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方式中，所述工程化Cas12i核酸酶包含如下突变组合：
E176R+K238R+T447R+E563R+N164Y+E323R+D362R，其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。该突变体以下被命名为CasXX，其序列编号为SEQ ID NO：8。
在一些实施方式中，所述工程化Cas12i核酸酶包含任一个下述突变组合：
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+R857A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+R719A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+K394A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+K844A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+R719A+K844A；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+R857A+K844A；其中，氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。这些Cas12i突变体是在CasXX(SEQ ID NO：8)基础上进行了进一步的优化所得到的优化的突变体。
在一些实施方式中，所述工程化Cas12i核酸酶包含如下突变组合：
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+K394A，其中，氨基酸位置编号 如SEQ ID NO：1所示的相应氨基酸位置所定义。该突变体以下被命名为“HF-20”，其序列如SEQ ID NO：20所示。
在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、E323R、和D362R突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、和I926R突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、E323R、和D362R突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、E323R、D362R、和I926G突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G、和L439(L+GG)突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G、和L439(L+G)突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、和D958R突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、I926R、和D958R突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、E323R、D362R、和D958R突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、I926R、E323R、D362R、和D958R突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G、和D958R突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G、L439(L+GG)、和D958R突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、E323R、D362R和K844A突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、E323R、D362R、R719A和K844A突变。在一些实施方式中，所述工程化Cas12i核酸酶包含E176R、K238R、T447R、E563R、N164Y、E323R、D362R、R857A和K844A突变。其中，所述氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
在一些实施方案中，为实现改善基因编辑效率的目的，也可以使用与上述工程化的Cas12i核酸酶具有至少约80％(例如任一至少约81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的工程化的Cas12i核酸酶。
在一些实施方案中，提供了一种工程化的Cas12i核酸酶，其包含如SEQ ID NOs： 2～24所示的任一氨基酸序列，或具有与SEQ ID NOs：2～24所示的任一氨基酸序列具有至少约80％(例如任一至少约81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的氨基酸序列。
参比Cas12i核酸酶
在一些实施方案中，所述参比Cas12i核酸酶为Cas12i1、Cas12i2，或其直系同源物。在一些实施方案中，所述参比Cas12i核酸酶为天然Cas12i1，或其变体(如自然存在的变体)。在一些实施方案中，所述参比Cas12i核酸酶为天然Cas12i2(如SEQ ID NO：1所示)，或其变体(如自然存在的变体)。在一些实施方案中，所述参比Cas12i核酸酶为经过工程改造后的Cas12i核酸酶(如本发明所述的包含(1)-(5)节中任一个或者多个突变的工程化的Cas12i核酸酶)。在一些实施方案中，所述参比Cas12i核酸酶为CasXX(SEQ ID NO：8)。
V-I型CRISPR-Cas12i已被鉴定为RNA-指导的DNA核酸内切酶系统。与诸如Cas12b或Cas9的CRISPR-Cas系统不同，基于Cas12i的CRISPR系统不需要tracrRNA序列。在一些实施方案中，所述RNA指导序列包括crRNA。通常，本文所述的crRNA包括直接重复序列和间隔区序列。在某些实施方案中，所述crRNA包括与指导序列或间隔区序列连接的直接重复序列、基本由或由其组成。在一些实施方案中，所述crRNA包括直接重复序列、间隔区序列和直接重复序列(DR-间隔区-DR)，其是其他CRISPR系统中的前体crRNA(pre-crRNA)构型的典型特征。在一些实施方案中，所述crRNA包括截短的直接重复序列和间隔区序列，其是经加工的或成熟的crRNA的典型特征。在一些实施方案中，所述CRISPR-Cas12i效应蛋白与RNA指导序列形成复合物，并且所述间隔区序列将复合物引导至与靶核酸的序列特异性结合，所述靶核酸与间隔区序列互补(如至少70％互补)。
在一些实施方案中，本申请的工程化的Cas12i是核酸内切酶，其结合靶序列的特定位点并在指导RNA的指导下切割，并且具有DNA和RNA内切核酸酶活性。在一些实施方案中，所述Cas12i能够通过加工前体crRNA阵列来进行自主的crRNA生物发生。自主的前体crRNA处理可促进Cas12i的递送，从而实现双切口应用，因为可以由单个crRNA转录物靶向两个单独的基因组位点。然后，Cas12i蛋白将CRISPR阵列加工成两个同源的crRNA，从而形成配对的切口复合物。V-I型(Cas12i)效应蛋白的复用(Multiplexing)是利用所述效应蛋白的前体crRNA处理能力完成的，其中可以针对具有不同序列的多个靶标在单个RNA指导序列上编程。这样，可以同时操纵多个基因或DNA靶标以用于治疗应用。在一些实施方案中，所述指导RNA包含由CRISPR阵列表达的前 体crRNA，所述CRISPR阵列由与未加工的DR序列交错的靶序列组成，通过所述效应蛋白的内在前体crRNA加工而重复以使得能够同时靶向一个、两个或多个位点。
来自多种生物体的Cas12i核酸酶可以用作所述参比Cas12i核酸酶，以提供本申请的工程化的Cas12i核酸酶及效应蛋白。示例性Cas12i核酸酶已经描述于例如WO2019/201331A1和US2020/0063126A1中，其通过整体引用而并入本文。在一些实施方案中，所述参比Cas12i核酸酶具有酶活性。在一些实施方案中，所述参比Cas12i是核酸酶，即切割靶双螺旋核酸(例如，双螺旋DNA)的两条链。在一些实施方案中，所述参比Cas12i是切口酶，即切割靶双螺旋核酸(例如，双螺旋DNA)的单链。在一些实施方案中，所述参比Cas12i核酸酶是酶失活的。在一些实施方案中，所述参比Cas12i核酸酶是Cas12i1、Cas12i2或Cas12i-Phi。在一些实施方案中，所述参比Cas12i核酸酶含有SEQ ID NO：1的序列。与Cas12i(如Cas12i2)或其功能衍生物具有一定序列同一性(例如至少约60％、70％、80％、85％、90％、95％、96％、97％、98％、99％或更多中任一个)的直系同源物可以用作设计本申请的工程化的Cas12i核酸酶或效应蛋白的基础。
工程化的Cas12i核酸酶的功能变体
在一些实施方案中，所述工程化的Cas12i核酸酶是基于天然存在的Cas12i核酸酶的功能变体(或功能衍生物)。在一些实施方案中，当与相应的工程化的Cas12i核酸酶(如包含上述(1)-(7)中任何一种或多种突变的工程化的Cas12i核酸酶)的氨基酸序列相比时，功能变体(或功能衍生物)的氨基酸序列具有至少一个氨基酸残基的不同(例如，具有缺失、插入、替换和/或融合)。在一些实施方案中，所述功能变体具有一个或多个突变，如氨基酸替换、插入和缺失。举例来说，与野生型天然存在的Cas12i核酸酶相比，或与前述工程化的Cas12i核酸酶相比，所述功能变体可包含1、2、3、4、5、6、7、8、9、10个或更多个氨基酸中任一个替换。在一些实施方案中，所述一个或多个替换是保守替换。在一些实施方案中，所述功能变体具有天然存在的Cas12i核酸酶的所有结构域。在一些实施方案中，所述功能变体不具有天然存在的Cas12i核酸酶的一个或多个结构域。在一些实施方案中，所述功能变体具有工程化的Cas12i核酸酶的所有结构域。在一些实施方案中，所述功能变体不具有工程化的Cas12i核酸酶的一个或多个结构域。在一些实施方式中，Cas12i核酸酶的功能变体的生物学活性由于其氨基酸的变化而变化，例如由天然核酸酶转变为酶失活突变体。
对于本文所述Cas12i变体蛋白(例如，切口酶Cas12i蛋白、失活或催化失活的Cas12i(dCas12i))中任一种，所述Cas12i变体可包括具有上述相同参数(例如，存在的结 构域、同一性百分比等)的Cas12i蛋白序列。
在一些实施方式中，所述工程化的Cas12i核酸酶的功能变体具有酶活性(如DNA双链或单链切割活性)，或至少具有参比Cas12i核酸酶(或其亲本工程化的Cas12i核酸酶)约60％(比如至少约65％、70％、80％、90％、95％，96％、97％、98％、99％、或100％中的任一个)的酶活性。在一些实施方式中，所述工程化Cas12i核酸酶的功能变体的酶活性是其亲本工程化Cas12i核酸酶的至少1.1倍(比如至少1.2倍、1.5倍、2倍、3倍、4倍、5倍、10倍或更多)的酶活性。
在一些实施方案中，所述工程化Cas12i核酸酶的功能变体与其工程化的Cas12i核酸酶的非功能变体突变形式相比，具有不同的催化活性。在一些实施方案中，所述功能变体突变(例如，氨基酸替换、插入和/或缺失)在Cas12i核酸酶的催化结构域(例如，RuvC结构域)中。在一些实施方案中，所述工程化Cas12i核酸酶的功能变体包含一个或多个催化结构域中的突变。切割双链靶核酸的一条链而不切割另一条链的Cas12i核酸酶，在本文中被称为“切口酶”(例如“Cas12i切口酶”)。在本文中，基本上不具有核酸酶活性的Cas12i核酸酶称为失活Cas12i蛋白(“dCas12i”)(与其融合的异源多肽(融合伴侣)可以提供核酸酶活性，详见下面“工程化的Cas12i效应蛋白”章节)。在一些实施方案中，相对于其非功能变体的突变形式而言，当功能变体突变酶的DNA切割活性小于约25％、10％、5％、1％、0.1％、0.01％或更低时，认为该Cas12i核酸酶功能变体基本上缺乏所有的DNA切割活性。
Cas12i核酸酶活性位点中一个或多个氨基酸残基的突变会导致降低或失去酶活的Cas12i(dCas12i)，在本发明中也称为“核酸酶活性缺失的Cas12i”或“酶失活突变体”。在一些实施方案中，本文提供的工程化的Cas12i核酸酶可以被修饰以具有减少或缺失的核酸酶活性，例如，核酸酶活性缺失的Cas12i与野生型Cas12i核酸酶(或其亲本工程化的Cas12i核酸酶)相比，核酸酶活性(如DNA双链或单链切割活性)降低至少约50％(比如降低至少约60％、70％、80％、90％、95％，96％、97％、98％、99％、或100％中的任一个)。所述Cas12i核酸酶活性可以通过几种方法来降低，例如，将突变引入所述Cas12i核酸酶的一个或多个结构域中：与PAM相互作用的结构域、参与打开DNA双链的结构域、RuvC结构域、与核酸(DNA/RNA)相互作用的区域等。在一些实施方案中，所述Cas12i核酸酶活性的催化残基(如通过任何通用鉴定方法鉴定出的催化残基)可以被不同的氨基酸残基(例如，甘氨酸或丙氨酸)替换以降低所述核酸酶活性。Cas12i1(如SEQ ID NO：13所示)的此类突变的实例包括D647A、E894A和/或D948A。Cas12i2(如SEQ ID NO：1所示)的此类突变的实例包括D599A、E833A、S883A、H884A、D886A、R900A和/或 D1019A。在一些实施方案中，所述工程化的Cas12i核酸酶或其功能衍生物(比如包含上述(1)-(7)中任何一种或多种突变的工程化的Cas12i核酸酶)包含以下一种或多种的核酸酶活性缺失突变：D599A、E833A、S883A、H884A、R900A和D1019A；其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
工程化的Cas12i核酸酶或其功能变体的特性
在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)与所述参比Cas12i核酸酶相比具有增加的活性。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)与所述参比Cas12i核酸酶相比具有降低的活性。在一些实施方案中，所述活性是靶DNA结合活性。在一些实施方案中，所述活性是位点特异性核酸酶活性。在一些实施方案中，所述活性是打开DNA双链的活性。在一些实施方案中，所述活性是双链DNA切割活性。在一些实施方案中，所述活性是单链DNA切割活性，包括例如位点特异性DNA切割活性或非特异性DNA切割活性。在一些实施方案中，所述活性是单链RNA切割活性，例如位点特异性RNA切割活性或非特异性RNA切割活性。在一些实施方案中，所述活性是在体外测量的。在一些实施方案中，所述活性是在细胞中测量的，如细菌细胞、植物细胞或真核细胞。在一些实施方案中，所述活性是在哺乳动物细胞如啮齿动物细胞或人细胞中测量的。在一些实施方案中，所述活性是在人细胞诸如293T细胞中测量的。在一些实施方案中，所述活性是在小鼠细胞，例如Hepa1-6细胞中测量的。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)具有与参比Cas12i核酸酶相比增加至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、1倍、1.1倍、1.5倍、2倍、3倍、4倍、5倍、10倍或更多中任一个的活性(如以上所述一种或多种活性，比如位点特异性核酸酶活性)。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)具有与参比Cas12i核酸酶相比降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、1倍、1.1倍、1.5倍、2倍、3倍、4倍、5倍、10倍或更多中任一个的活性(如以上所述一种或多种活性，比如位点特异性核酸酶活性)。所述工程化的Cas12i核酸酶(或其功能变体)的位点特异性核酸酶活性可以使用本领域中已知的方法来测量，包括例如，凝胶迁移测定，如本文提供的实施例中所述的基于琼脂糖凝胶电泳的体外切割测定。
在一些实施方案中，所述活性是细胞中的基因编辑活性。在一些实施方案中，所述细胞是细菌细胞、植物细胞或真核细胞。在一些实施方案中，所述细胞是哺乳动物细胞如啮齿动物细胞或人细胞。在一些实施方案中，所述细胞是293T细胞。在一些实施方案 中，所述活性是在小鼠细胞，例如Hepa1-6细胞中测量的。在一些实施方案中，所述活性是在细胞中靶基因组位点的插入缺失形成活性，例如通过所述工程化的Cas12i核酸酶(或其功能变体)对靶核酸进行位点特异性切割和通过非同源末端连接(NHEJ)机制进行DNA修复。在一些实施方案中，所述活性是在细胞中靶基因组位点插入外源核酸序列的活性，例如通过所述工程化的Cas12i核酸酶(或其功能变体)对靶核酸进行位点特异性切割和通过同源重组(HR；通过进一步引入一个修复模板)机制进行DNA修复。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)与参比Cas12i核酸酶相比在细胞(例如人细胞如293T细胞，或小鼠Hepa1-6细胞)的靶基因组位点处增加至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、1倍、1.1倍、1.5倍、2倍、3倍、4倍、5倍、10倍或更多中任一个的基因编辑(例如，切割、插入缺失形成、或修复)活性。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)与参比Cas12i2核酸酶相比在细胞(例如人细胞如293T细胞或小鼠Hepa1-6细胞)的多个(例如2、3、4、5、6、7、8、9、10个或更多个)靶基因组位点处增加至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、1倍、1.1倍、1.5倍、2倍、3倍、4倍、5倍、10倍或更多中任一个的基因编辑(例如，切割、插入缺失形成、或修复)活性。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)与所述参比Cas12i核酸酶相比，能够编辑更多数目(例如2、3、4、5、6、7、8、9、10、20、30、40、50、60、70个或更多个)的基因组位点，比如能识别更多(例如2、3、4、5、6、7、8、9、10、20、30、40、50、60、70个或更多个)的PAM序列。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)的共有PAM序列与所述参比Cas12i核酸酶相同。
可使用本领域已知的方法确定体外或细胞中工程化的Cas12i核酸酶(或其功能变体)的基因编辑效率，包括例如T7核酸内切酶1(T7E1)测定、靶DNA的测序(包括例如，Sanger序列，以及二代测序)、由分解追踪插入缺失(TIDE)测定或通过扩增子分析进行插入缺失检测(IDAA)测定。参见例如Sentmanat MF et al.，“A survey of validation strategies for CRISPR-Cas9 editing，”Scientific Reports，2018，8，文章编号888，其通过整体引用而并入本文。在一些实施方案中，例如，如本文实施例中所述，使用靶向的二代测序(NGS)来测量细胞中所述工程化的Cas12i核酸酶的基因编辑效率。可用于确定所述工程化的Cas12i核酸酶(或其功能变体)的基因编辑效率的示例性基因组位点包括但不限于CCR5、AAVS、CD34、RNF2和EMX1。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)的基因编辑效率为所述工程化的Cas12i核酸酶在至少5个、10个、15个、20个、25个、30个、35个、40个、45个、50个、55个、60个、65个或更多位点(如人类 细胞基因组位点)的平均基因编辑效率。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)的基因编辑效率(例如插入缺失率)达到至少10％、20％、30％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或更高。
在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)与所述参比Cas12i核酸酶相比具有增加的靶向特异性，具有降低的脱靶率(例如减少识别的脱靶位点、和/或降低对一个或多个脱靶位点的编辑效率)，和/或增加的靶序列编辑效率。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)与所述参比Cas12i核酸酶相比，降低至少约5％(如降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％中任一个)的脱靶率。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)与所述参比Cas12i核酸酶相比，减少至少一个(如至少2、3、4、5、6、7、8、9、10、20、50或更多)脱靶位点。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)与所述参比Cas12i核酸酶相比，对靶序列的编辑效率相同或相似(比如在1.1倍以内)，或对靶序列的编辑效率增加(例如是参比Cas12i核酸酶至少1.2倍、1.5倍、2倍、3倍、5倍、10倍或更多)。在一些实施方案中，所述工程化的Cas12i核酸酶(或其功能变体)与所述参比Cas12i核酸酶相比，降低至少约5％的脱靶率，且对靶序列的编辑效率相同、相似(比如在1.1倍以内)、或增加。
指导RNA或crRNA
在一些实施例中，指导RNA或crRNA从5’到3’包括以下或由以下组成：直接重复序列、间隔序列。在一些实施例中，指导RNA从5’到3’包括以下或由以下组成：直接重复序列、间隔序列、直接重复序列的串联构建的核苷酸序列。在一些实施例中，RNA指导物包括crRNA。在一些实施例中，指导RNA不包含tracrRNA。
通常，本文描述的crRNA包括直接重复序列和间隔子序列。在某些实施例中，crRNA包括以下，基本上由以下组成或由以下组成：连接到指导序列或间隔子序列的直接重复序列。在一些实施例中，crRNA包括直接重复序列、间隔子序列和直接重复序列(DR-间隔子-DR)，其是其他CRISPR系统中典型的前体crRNA(前crRNA)构型。在一些实施例中，crRNA包括截短的直接重复序列和间隔子序列，其是典型的经加工或成熟的crRNA。在一些实施例中，CRISPR-Cas效应子蛋白与RNA指导物形成复合物，间隔子序列将该复合物导向与跟间隔子序列互补的靶核酸进行序列特异性结合。
在一些实施例中，RNA指导物包含直接重复。在一些实施例中，RNA指导物可以形成二级结构，例如，如本文所述的茎环结构。
在一些实施例中，本文描述的CRISPR系统包括多个RNA指导物(例如，2，3，4，5， 10，15个或更多个)或编码多个RNA指导物的多个核酸。在一些实施例中，本文描述的CRISPR系统包括单个RNA链或编码单个RNA链的核酸，其中RNA指导物串联布置。所述单个RNA链可包括相同RNA指导物的多个拷贝、不同RNA指导物的多个拷贝、或其组合。在一些实施方式中，每种RNA指导物对不同的靶核酸具有特异性。
在一些实施例中，本文描述的CRISPR系统包括RNA指导物或编码RNA指导物的核酸。在一些实施例中，RNA指导物包含以下或由以下组成：直接重复序列和能够与靶核酸杂交(例如，在适当条件下杂交)的间隔子序列。
在一些实施方式中，RNA指导序列可以允许形成CRISPR效应子复合物并成功结合靶序列，同时不允许成功的核酸酶活性(即，没有核酸酶活性/没有引起插入缺失)的方式进行修饰。这些经修饰的指导物序列被称为“失活指导物”或“失活指导物序列”。这些失活指导物或失活指导物序列对于核酸酶活性而言可以是催化上失活的或构象上失活的。失活指导物序列通常比导致活性RNA切割的相应指导物序列短。在一些实施例中，失活指导物比具有核酸酶活性的相应RNA指导物短至少约5％、10％、20％、30％、40％或50％。RNA指导物的失活指导物序列的长度可以是13至15个核苷酸(例如，13、14或15个核苷酸)、15至19个核苷酸、或17至18个核苷酸(例如，17个核苷酸)。在一些实施方式中，失活指导物RNA能够与靶序列杂交，使得CRISPR系统被导向细胞中目的基因组基因座而没有可检测的切割活性。
本文描述的RNA指导物和crRNA的序列和长度可以优化。在一些实施例中，RNA指导物的优化长度可通过鉴定crRNA的加工形式或通过crRNA的RNA指导物的经验长度研究来确定。在一些实施方式中，RNA指导序列包含碱基修饰。
在一些实施方式中，本发明还提供核酸(例如cDNA)的所有可能的变体，所述变体可以通过基于可能的密码子选择来选择组合而制备。这些组合是根据应用于编码天然存在变体的多核苷酸的标准三联体遗传密码进行的，并且所有这些变体都被认为是具体公开的。
间隔(spacer)序列
在一些实施例中，间隔序列(或间隔子、指导序列)可与靶核酸(如DNA)的靶序列互补，例如至少约70％(例如至少约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)互补。在一些实施例中，间隔序列与靶核酸(如DNA)的靶序列有至少15个(如至少16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45、50或更多个)核苷酸互补。
在本领域已知，不需要完全的互补性，前提是有足够的互补性发挥作用。可以通过 引入错配(例如一个或多个错配，例如间隔子序列和靶序列之间(包括沿着间隔子/靶的错配的位置)的1或2个错配)来利用切割效率的调节。错配，例如双错配，位于越中心(即，不在3’或5’端)；切割效率受到的影响越大。因此，通过选择沿间隔子序列的错配位置，可以调节切割效率。例如，如果期望靶的小于100％的切割(例如，在细胞群体中)，则间隔子序列中可引入间隔子序列和靶序列之间的1或2个错配。
所述指导序列可以具有合适的长度。RNA指导物的间隔子长度可在约11至50个(例如约15至50个)核苷酸的范围内。在一些实施方案中，所述指导序列在约18至约35个核苷酸之间，包括例如18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35个核苷酸。在一些实施例中，RNA指导物的间隔子长度为至少16个核苷酸、至少17个核苷酸、至少18个核苷酸、至少19个核苷酸、至少20个核苷酸、至少21个核苷酸或至少22个核苷酸。在一些实施例中，间隔子长度为15至17个核苷酸、15至23个核苷酸、16至22个核苷酸、17至20个核苷酸、20至24个核苷酸(例如、20、21、22、23或24个核苷酸)、23至25个核苷酸(例如、23、24或25个核苷酸)、24至27个核苷酸、27至30个核苷酸、30至45个核苷酸(例如、30、31、32、33、34、35、40或45个核苷酸)、30或35至40个核苷酸、41至45个核苷酸、45至50个核苷酸，或更长。在一些实施例中，RNA指导物的间隔子长度为31个核苷酸。在一些实施例中，RNA指导物的直接重复长度为至少21个核苷酸，或为21至37个核苷酸(例如，23、24、25、30、35或36个核苷酸)。在一些实施例中，间隔序列包括以下或由以下组成：约15至约34个核苷酸(例如，16、17、18、19、20、21或22个核苷酸)。在一些实施例中，间隔序列长度在17个核苷酸和31个核苷酸之间。在一些实施例中，间隔序列长度在15个核苷酸和24个核苷酸之间。在一些实施例中，RNA指导物的直接重复长度为23或20个核苷酸。
直接重复(DR)序列
直接重复序列能够引导Cas12i蛋白(如任一本发明所述工程化的Cas12i核酸酶、其功能变体或效应蛋白)结合至指导gRNA(或crRNA)以形成靶向靶序列的CRISPR-Cas复合物。任何能够引导本申请中工程化的Cas12i核酸酶或效应蛋白结合至指导gRNA(或crRNA)以形成靶向靶序列的CRISPR-Cas复合物的DR都可以用于本发明，例如US11168324(其全部内容通过整体引用并入本文)中描述的DR序列。
直接重复可以包括两个核苷酸段，它们可以彼此互补，被间插的核苷酸隔开，这样使得直接重复可以杂交形成双链RNA双链体(dsRNA双链体)，导致茎环结构，其中两个互补核苷酸段形成茎，并且间插的核苷酸形成环或发夹。例如，形成“环”的中间核苷酸 具有从约6个核苷酸到约8个核苷酸，或约7个核苷酸的长度。在不同的实施例中，茎可以包括至少2个、至少3个、至少4个或5个碱基对。
在一些实施方式中，直接重复可以包括长度为约4个至约7个(如4、5、6、7个)核苷酸的两个核苷酸互补段，其间间隔约5个至约9个(如5、6、7、8、9个)核苷酸。本领域技术人员可以模拟已知的直接重复的结构。
直接重复可以包括以下或由以下组成：约13至约23个核苷酸、约22至约40个核苷酸、或约23至约38个核苷酸或约23至约36个核苷酸。
在一些实施例中，直接重复序列包括靠近3’末端(紧邻间隔子序列)的茎环结构。在一些实施例中，直接重复序列包括靠近3’末端的茎环，其中茎的长度为5个核苷酸。在一些实施例中，直接重复序列包括靠近3’末端的茎环，其中茎的长度为5个核苷酸，并且环的长度为7个核苷酸。在一些实施例中，直接重复序列包括靠近3’末端的茎环，其中茎的长度为5个核苷酸，并且环的长度为6、7或8个核苷酸。
在一些实施例中，直接重复序列包括靠近3’末端的序列5’-CCGUCNNNNNNUGACGG-3’(SEQ ID NO：68)，其中N是指任何核碱基。在一些实施例中，直接重复序列包括靠近3’末端的序列5’-GUGCCNNNNNNUGGCAC-3’(SEQ ID NO：69)，其中N是指任何核碱基。
在一些实施例中，直接重复序列包括靠近3’末端的序列5’-GUGUCN 5-6UGACAX 1-3’(SEQ ID NO：70或71)，其中N 5-6是指任何5或6个核碱基的连续序列，并且X 1是指C或T或U。在一些实施例中，直接重复序列包括靠近3’末端的序列5’-UCX 3UX 5X 6X 7UUGACGG-3’(SEQ ID NO：72)，其中X 3是指C或T或U，X 5是指A或T或U，X 6是指A或C或G，并且X 7是指A或G。在一些实施例中，直接重复序列包括靠近3’末端的序列5’-CCX 3X 4X 5CX 7UUGGCAC-3’(SEQ ID NO：73)，其中X 3是指C或T或U，X 4是指A或T或U，X 5是指C或T或U，并且X 7是指A或G。在一些实施例中，编码直接重复序列的核苷酸包括以下或由以下组成：与SEQ ID NO：59(AGAAATCCGTCTTTCATTGACGG)或SEQ ID NO：79(GTTGCAAAACCCAAGAAATCCGTCTTTCATTGACGG)至少约80％相同，例如至少约85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％相同的核苷酸序列。在一些实施例中，编码直接重复序列的核苷酸包含SEQ ID NO：79的至少21个(例如21、22、23、24、25、26、27、28、29、30、31、32、33、34、35或36个)核苷酸。
“茎环结构”是指具有二级结构的核酸，所述二级结构包括已知或预测形成双链(茎部分)的核苷酸区域，所述双链(茎部分)在一侧由主要为单链核苷酸的区域(环部分)连接。术 语“发夹”和“折回”结构在本文中也用于指茎环结构。这样的结构在本领域中是公知的，并且这些术语与其在本领域中的公知含义一致地使用。如本领域已知的，茎环结构不需要精确的碱基配对。因此，茎可以包括一个或多个碱基错配。可替代地，碱基配对可以是精确的，即不包括任何错配。在一些实施例中，直接重复的茎由5个互相杂交的互补核碱基组成，并且环长度是6、7或9个核苷酸。
在一些实施例中，编码直接重复的序列包含以下或由以下组成：SEQ ID NO：79所示序列。在一些实施例中，编码直接重复的序列包含以下或由以下组成：SEQ ID NO：79核酸序列的具有起始三个5’核苷酸的截短的核酸。在一些实施例中，编码直接重复的序列包含以下或由以下组成：SEQ ID NO：79核酸序列的具有起始四个5’核苷酸的截短的核酸。在一些实施例中，编码直接重复的序列包含以下或由以下组成：SEQ ID NO：79核酸序列的具有起始五个5’核苷酸的截短的核酸。在一些实施例中，编码直接重复的序列包含以下或由以下组成：SEQ ID NO：79核酸序列的具有起始六个5’核苷酸的截短的核酸。在一些实施例中，编码直接重复的序列包含以下或由以下组成：SEQ ID NO：79核酸序列的具有起始七个5’核苷酸的截短的核酸。在一些实施例中，编码直接重复的序列包含以下或由以下组成：SEQ ID NO：79核酸序列的具有起始八个5’核苷酸的截短的核酸。在一些实施例中，编码直接重复的序列包含以下或由以下组成：SEQ ID NO：59所示序列。
在一些实施方式中，直接重复是SEQ ID NO：59或79编码的RNA序列的“功能性变体”，例如“功能性截短版本”、“功能性延长版本”、或“功能性替换版本”，比如SEQ ID NO：79的一部分(截短版本)，仍然具有DR功能。DR“功能性变体”是参比DR(如亲本DR)的5’和/或3’端延长(功能性延长版本)或截短(功能性截短版本)，和/或参比DR序列中插入、缺失、和/或替换(功能性替换版本)一个或多个核苷酸后，依然具有参比DR的至少20％(如至少约任何30％、40％、50％、60％、70％、80％、90％、95％、或更高)功能的DR序列，即介导Cas12i蛋白与相对应的crRNA结合的功能。DR功能性变体一般保留可供Cas12i蛋白结合的茎环样二级结构或其部分。在一些实施方式中，DR或其功能性变体包含一个可供Cas12i蛋白结合的茎环样二级结构或其部分。在一些实施方式中，DR或其功能性变体包含至少两个(比如2、3、4、5或更多个)可供Cas12i蛋白结合的茎环样二级结构或其部分。
引导编辑指导prime editing guide RNA(pegRNA)
PEgRNA(相对于标准向导RNA)改变为包含延伸部分，该延伸部分提供编码单链DNA瓣的DNA合成模板序列，其与待编辑的靶向内源性DNA序列的链同源但包含期望 的一个或多个核苷酸变化，并且在被聚合酶(如，逆转录酶)合成之后掺入靶DNA分子中。PEgRNA已描述于例如WO2020191246、WO2021226558，其通过整体引用而并入本文。
在不同实施方案中，延伸的向导RNA包含(a)向导RNA和(b)在向导RNA的5′或3′端处或在向导RNA的分子内位置中的RNA延伸。优选地，延伸部分的分子内定位不破坏原间隔区的功能。RNA延伸可包含(i)包含期望的核苷酸变化的逆转录模板序列，(ii)逆转录引物结合位点，和(iii)任选的接头序列。在不同实施方案中，逆转录模板序列可编码与邻近切口位点的内源性DNA序列互补的单链DNA瓣，其中单链DNA瓣包含期望的核苷酸变化。单链DNA瓣可在切口位点置换内源性单链DNA。在不同实施方案中，掺入靶DNA中的期望的核苷酸变化可以是单核苷酸变化(如，转换或颠换)、一个或多个核苷酸的插入、或者一个或多个核苷酸的缺失。
在不同实施方案中，期望的核苷酸变化可以是单核苷酸取代(如，转换或颠换变化)、缺失或插入。例如，期望的核苷酸变化可以是(1)G至T取代，(2)G至A取代，(3)G至C取代，(4)T至G取代，(5)T至A取代，(6)T至C取代，(7)C至G取代，(8)C至T取代，(9)C至A取代，(10)A至T取代，(11)A至G取代，或(12)A至C取代。
PAM
本发明所述的工程化的Cas12i核酸酶(或其功能变体)能够识别原间隔区相邻基序(PAM)。在一些实施方式中，所述靶核酸包括PAM。在一些实施方式中，PAM位于靶核酸中与引导RNA的靶向序列互补的序列的5‘端。在一些实施方式中，PAM包括核酸序列5′-TTN-3′、5′-TTH-3′、5′-TTY-3′或5′-TTC-3′，或者由其组成。在一些实施方式中，适用于本发明所述的工程化的Cas12i核酸酶(或其功能变体)的PAM包括核酸序列5′-N NNN-3′( N＝A，T，G，C)或者由其组成，比如5’-NTTN-3’，5’-NTAN-3’，5’-NTCN-3’，5’-NTGN-3’，5’-NATN-3’，5’-NAAN-3’，5’-NACN-3’，5’-NAGN-3’，5’-NCTN-3’，5’-NCAN-3’，5’-NCCN-3’，5’-NCGN-3’，5’-NGTN-3’，5’-NGAN-3’，5’-NGCN-3’，5’-NGGN-3’。在一些实施方式中，所述PAM包括核酸序列5′-TTTN-3′或者由其组成。在一些实施方式中，所述PAM包括核酸序列5′-TTN-3′或者由其组成。
在一些实施方式中，适用于本发明所述的工程化的Cas12i核酸酶(或其功能变体)的PAM包括核酸序列5′-TTTA-3′、5′-CTTA-3′、5′-GTTA-3′、5′-ATTA-3′、5′-TTTC-3′、5′-CTTC-3′、5′-GTTC-3′、5′-ATTC-3′、5′-TTTG-3′、5′-CTTG-3′、5′-GTTG-3′、5′-ATTG-3′、5′-TTTT-3′、5′-CTTT-3′、5′-GTTT-3′、5′-ATTT-3′。
工程化的Cas12i效应蛋白
本申请提供了工程化的Cas12i(如Cas12i2)效应蛋白，其包含本发明所述任何工程化的Cas12i核酸酶或其功能变体(例如SEQ ID NO：8所示CasXX)，其具有改善的活性，例如靶标结合、双链切割活性、切口酶活性和/或基因编辑活性。在一些实施方案中，提供了工程化的Cas12i效应蛋白(例如，Cas12i核酸酶、Cas12i切口酶、Cas12i融合效应蛋白或分割型(split)Cas12i效应蛋白)，其包含本文所述工程化的Cas12i核酸酶或其功能衍生物(如dCas12i)中的任一种。在一些实施方案中，工程化的Cas12i效应蛋白包含本发明所述任何工程化的Cas12i核酸酶或其功能变体，或主要由其组成、或由其组成。
还提供了基于本文所述工程化的Cas12i2核酸酶或其功能变体(例如SEQ ID NO：8所示CasXX)中任一种的工程化的Cas12i效应蛋白。在一些实施方案中，所述工程化的Cas12i效应蛋白具有酶活性(如DNA双链切割活性)。在一些实施方案中，所述工程化的Cas12i效应蛋白有切割靶双螺旋核酸(例如，双螺旋DNA)的两条链的核酸酶活性。在一些实施方案中，所述工程化的Cas12i效应蛋白有切口酶活性，即切割靶双螺旋核酸(例如，双螺旋DNA)的单链。在一些实施方案中，所述工程化的Cas12i效应蛋白包含所述工程化的Cas12i核酸酶的酶失活突变体。
分割型Cas12i效应蛋白
本申请还提供了基于本文所述工程化的Cas12i核酸酶或其功能变体(例如SEQ ID NO：8所示CasXX)中任一种的分割型Cas12i效应蛋白。分割型Cas12i效应蛋白对于递送可能是有利的。在一些实施方案中，所述工程化的Cas12i效应蛋白被分割成酶的两个部分，可以将它们重构在一起以提供基本起作用的Cas12i效应蛋白。可利用已知方法提供Cas效应蛋白，例如，Cas12和Cas9蛋白的分割形式已描述于例如WO2016/112242、WO2016/205749和PCT/CN 2020/111057，其通过整体引用而并入本文。
在一些实施方案中，提供了分割型Cas12i效应蛋白，其包含第一多肽和第二多肽，所述第一多肽包含本文所述工程化的Cas12i核酸酶或其功能衍生物中任一种的N末端部分，所述第二多肽包含所述工程化的Cas12i核酸酶或其功能衍生物中任一种的C末端部分，其中所述第一多肽和第二多肽能够在包含指导序列的指导RNA存在下彼此缔合，以形成与靶核酸特异性结合的CRISPR复合物，所述靶核酸包含与所述指导序列互补的靶序列。在一些实施方案中，所述第一多肽和第二多肽各自包含二聚化结构域。在一些实施方案中，所述第一二聚化结构域和第二二聚化结构域在诱导剂(例如雷帕霉素)存在下彼此缔合。在一些实施方案中，所述第一多肽和第二多肽不包含二聚化结构域。在一些实施方案中，所述分割型Cas12i效应蛋白是自诱导的。
本发明所述工程化的Cas12i效应蛋白可以以不影响催化结构域的方式进行分割。Cas12i效应蛋白可以用作核酸酶(包括切口酶)或可以是灭活的酶，其本质上是具有很少或没有催化活性(例如由于其催化结构域中的突变)的RNA指导的DNA结合蛋白。
在一些实施方案中，工程化的Cas12i效应蛋白的核酸酶叶和α-螺旋叶表达为分开的多肽。尽管所述核酸酶叶和α-螺旋叶不自行相互作用，但RNA指导序列将它们募集到一个复合物中，所述复合物概括了全长Cas12i核酸酶的活性并催化位点特异性的DNA切割。在一些实施方案中，经修饰的RNA指导序列可以用于通过防止二聚化而消除分割型酶的活性，从而允许诱导型二聚化系统的发展。所述分割型酶描述于例如，Wright，Addison V.，et al.“Rational design of a split-Cas9 enzyme complex，”Proc.Nat′l.Acad.Sci.，112.10(2015)：2984-2989，其通过整体引用并入本文。
本文所述分割型Cas12i效应蛋白部分可设计为通过将参比工程化的Cas12i效应蛋白(例如全长工程化的Cas12i核酸酶)在一个分割位置处分开(即，分割)成两半，所述位置是所述参比Cas12i效应蛋白的N端部分与C端部分分开的点。在一些实施方案中，所述N末端部分包含所述参比Cas12i效应蛋白氨基酸残基1至X，而所述C末端部分包含氨基酸残基X+1至所述参比Cas12i效应蛋白的C末端。在该实例中，编号是连续的，但这并非必需的，因为还考虑了氨基酸(或编码它们的核苷酸)可以从分割的末端和/或突变(例如，插入、删除和替换)中任一个在所述多肽链的内部区域修剪而来，条件是重构的工程化Cas12i效应蛋白保留了足够的DNA结合活性(如果需要)、DNA切口酶或切割活性，例如与所述参比Cas12i效应蛋白相比，具有至少约40％(如至少约50％、60％、70％、80％、90％、95％或更高)的活性。
可以通过计算机(in silico)设计分割点并将其克隆到构建体中。在此过程中，可以将突变引入分割型Cas12i效应蛋白，并且可以去除非功能结构域。在一些实施方案中，所述分割型Cas12i效应蛋白的两个部分或片段(即，N末端和C末端片段)可以形成完整的Cas12i效应蛋白，其包含例如完整Cas12i效应蛋白序列的至少约70％(如至少约80％、90％、95％、96％、97％、98％、99％或更高)。
所述分割型Cas12i效应蛋白可各自包含一个或多个二聚化结构域。在一些实施方案中，所述第一多肽包含与第一分割型Cas12i效应蛋白部分融合的第一二聚结构域，并且所述第二多肽包含与第二分割型Cas12i效应蛋白部分融合的第二二聚结构域。可通过肽接头(例如，柔性肽接头如GS接头)或化学键将所述二聚化结构域融合至所述分割型Cas12i效应蛋白部分。在一些实施方案中，所述二聚化结构域与所述分割型Cas12i效应蛋白部分的N末端融合。在一些实施方案中，所述二聚化结构域与所述分割型Cas12i效 应蛋白部分的C末端融合。
在一些实施方案中，该分割型Cas12i效应蛋白不包含任何二聚化结构域。
在一些实施方案中，所述二聚化结构域促进两个分割型Cas12i效应蛋白部分的缔合。在一些实施方案中，所述分割型Cas12i效应蛋白部分被诱导剂诱导而缔合或二聚化为功能性Cas12i效应蛋白。在一些实施方案中，所述分割型Cas12i效应蛋白包含可诱导的二聚化结构域。在一些实施方案中，所述二聚化结构域不是可诱导的二聚化结构域，即，所述二聚化结构域在诱导剂不存在下进行二聚化。
诱导剂可以是除指导RNA(例如crRNA)以外的诱导能量源或诱导分子。所述诱导剂通过诱导的二聚化结构域的二聚化将两个分割型Cas12i效应蛋白部分重构为功能性Cas12i效应蛋白。在一些实施方案中，所述诱导剂通过可诱导的二聚化结构域的诱导缔合的作用将两个分割型Cas12i效应蛋白部分聚集在一起。在一些实施方案中，在没有诱导剂的情况下，两个分割型Cas12i效应蛋白部分不彼此缔合以重构为功能性Cas12i效应蛋白。在一些实施方案中，在没有诱导剂的情况下，两个分开的Cas12i效应蛋白部分可以在指导RNA(例如crRNA)存在下彼此缔合以重构为功能性Cas12i效应蛋白。
本申请的诱导剂可以是热、超声、电磁能或化合物。在一些实施方案中，所述诱导剂是抗生素、小分子、激素、激素衍生物、类固醇或类固醇衍生物。在一些实施方案中，所述诱导剂是脱落酸(ABA)、多西霉素(DOX)、异丙基苯甲酸(cumate)、雷帕霉素、4-羟基他莫昔芬(4OHT)、雌激素或蜕皮激素。在一些实施方案中，所述分割型Cas12i效应系统是选自下组的诱导剂控制的系统：基于抗生素的诱导系统、基于电磁能的诱导系统、基于小分子的诱导系统、基于核受体的诱导系统和基于激素的诱导系统。在一些实施方案中，所述分割型Cas12i效应系统是选自下组的诱导剂控制的系统：四环素(Tet)/DOX诱导系统、光诱导系统、ABA诱导系统、异丙基苯甲酸(cumate)阻遏物/操纵子系统、4OHT/雌激素诱导系统、基于蜕皮激素的诱导系统和FKBP12/FRAP(FKBP12-雷帕霉素复合物)诱导系统。这样的诱导剂也讨论于本文和PCT/US2013/051418，其通过整体引用而并入本文。FRB/FKBP/雷帕霉素系统已描述于Paulmurugan and Gambhir，Cancer Res，August 15，2005 65；7413，以及Crabtree et al.，Chemistry&Biology 13，99-107，Jan 2006，其通过整体引用而并入本文。
在一些实施方案中，成对的分割型Cas12i效应蛋白是分开的并且是无活性的，直到诱导了所述二聚化结构域的二聚化(例如，FRB和FKBP)，该二聚化导致功能性Cas12i效应蛋白核酸酶的再组装。在一些实施方案中，包含诱导型二聚体(例如FRB)的第一半部分的第一分割型Cas12i效应蛋白被分开递送，和/或处在与包含诱导型二聚体(例如 FKBP)的第二半部分的第二分割型Cas12i效应蛋白分开的位置。
可用于本文所述的诱导剂控制的分割型Cas12i效应系统中的其他示例性的基于FKBP的诱导系统包括但不限于：在FK506存在下与钙调神经磷酸酶(CNA)进行二聚化的FKBP；在FKCsA存在下与CyP-Fas进行二聚化的FKBP；在雷帕霉素存在下与FRB进行二聚化的FKBP；在香豆霉素存在下与GryB进行二聚化的GyrB；在赤霉素存在下与GID1进行二聚化的GAI；或在HaXS存在下与HaloTag进行二聚化的Snap-tag。
也考虑了FKBP家族本身内的替代方案。例如，在FK1012存在下FKBP进行均二聚(即，一个FKBP与另一种FKBP进行二聚化)。
在一些实施方案中，所述二聚化结构域是FKBP，而诱导剂是FK1012。在一些实施方案中，所述二聚化结构域是GryB，而诱导剂是香豆霉素。在一些实施方案中，所述二聚化结构域是ABA，而诱导剂是赤霉素。
在一些实施方案中，可以在不存在诱导剂的情况下自动诱导(即，自动激活或自诱导)所述分割型Cas12i效应蛋白部分以缔合/二聚化为功能性Cas12i效应蛋白。不受任何理论或假设的束缚，可以通过与指导RNA如crRNA结合来介导所述分割型Cas12i效应蛋白部分的自动诱导。在一些实施方案中，所述第一多肽和第二多肽不包含二聚化结构域。在一些实施方案中，所述第一多肽和第二多肽包含二聚化结构域。
在一些实施方案中，本文所述分割型Cas12i效应系统(包括诱导剂控制的和自动诱导的系统)的重构的Cas12i效应蛋白，相对于参比Cas12i效应蛋白编辑效率，具有至少约60％(如至少约70％、75％、80％、85％、90％、95％、98％、99％或更高中任一种)的编辑效率。
在一些实施方案中，本文所述诱导剂控制的分割型Cas12i效应系统的重构的Cas12i效应蛋白，在没有诱导剂存在下(即由于自动诱导)，相对于参比Cas12i效应蛋白编辑效率，其具有不超过约50％(如不超过约45％、40％、35％、30％、25％、20％、15％、10％、5％或更低效率中任一个)的编辑效率。
融合Cas12i效应蛋白
本申请还提供了工程化的Cas12i效应蛋白，其包含另外的蛋白结构域和/或组分，如接头、核定位/输出序列、功能结构域和/或报告蛋白。
在一些实施方案中，所述工程化的Cas12i效应蛋白是包含一个或多个异源蛋白结构域(例如，约或大于约1、2、3、4、5、6、7、8、9、10个或更多个结构域)以及任一本发明所述工程化的Cas12i核酸酶的核酸靶向结构域或其功能衍生物的蛋白复合物。在一些实施方案中，所述工程化的Cas12i效应蛋白是包含与所述工程化的Cas12i核酸酶或其功 能变体(例如SEQ ID NO：8所示CasXX)融合的一个或多个异源蛋白结构域(例如约或多于约1、2、3、4、5、6、7、8、9、10个或更多个结构域)的融合蛋白。
在一些实施方案中，本申请的工程化的Cas12i效应蛋白可包含(例如，通过融合蛋白，如通过一个或多个肽接头，例如GS肽接头等)一个或多个功能结构域或缔合(例如，通过多种蛋白的共表达)于它。在一些实施方案中，所述一个或多个功能结构域是酶结构域。这些功能结构域可以具有多种活性，例如DNA和/或RNA甲基化酶活性、核苷酸脱氨酶活性(如腺苷脱氨酶活性、胞苷脱氨酶活性)、脱甲基酶活性、转录激活活性、转录抑制活性、转录释放因子活性、组蛋白修饰活性、RNA切割活性、DNA切割活性(如双链内切酶活性、切口酶活性)、核酸结合活性和开关活性(例如，光诱导或化学诱导的)。在一些实施方案中，所述一个或多个功能结构域是转录激活结构域(即，反式激活结构域)或阻遏物结构域。在一些实施方案中，所述一个或多个功能结构域是组蛋白修饰结构域。在一些实施方案中，所述一个或多个功能结构域是转座酶结构域、HR(同源重组)机构结构域、重组酶结构域和/或整合酶结构域。在一些实施方案中，所述功能结构域是Krüppe1相关盒(KRAB)、VP64、VP16、Fok1、P65、HSF1、MyoD1、生物素-APEX、APOBEC1、AID、PmCDA1、Tad1和M-MLV逆转录酶。在一些实施方案中，所述功能结构域选自下组：翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域(例如，CBE或ABE结构域)、逆转录酶结构域、报告分子结构域(例如，荧光结构域)和核酸酶结构域。
在一些实施方式中，所述功能结构域具有修饰靶DNA或靶DNA相关蛋白的活性，所述活性选自核酸酶活性(例如，HNH核酸酶，RuvC核酸酶，Trex1核酸酶，Trex2核酸酶)、甲基化活性、脱甲基化活性、DNA修复活性、DNA损伤活性、脱氨基活性、歧化酶活性、烷基化活性、脱嘌呤活性、氧化活性、嘧啶二聚体形成活性、整合酶活性、转座酶活性、重组酶活性、聚合酶活性、连接酶活性、解旋酶活性、光裂合酶活性、糖基化酶活性、乙酰基转移酶活性、脱乙酰酶活性、激酶活性、磷酸酶活性、泛素连接酶活性、去泛素化活性、腺苷酸化活性、脱腺苷酸化活性、SUMO化活性、脱SUMO化活性、核糖基化活性、脱核糖基化活性、豆蔻酰化活性、脱豆蔻酰化活性、糖基化活性(例如，来自O-GlcNAc转移酶)、脱糖基化活性、转录抑制活性和转录激活活性中的一种或多种。靶DNA相关蛋白是指可结合靶DNA的蛋白，或可与可结合靶DNA的蛋白相结合的蛋白，比如组蛋白、转录因子、Mediator等。
在一些实施方案中，所述工程化的Cas12i效应蛋白中一个或多个功能结构域的定位允许功能结构域的正确空间定向以影响具有所赋予功能作用的靶标。例如，如果所述功 能结构域是转录激活子(例如，VP16、VP64或p65)，则将所述转录激活子放置在使其能够影响靶标转录的空间取向上。同样地，定位转录阻遏物以影响靶标的转录，并且将核酸酶(例如，Fok1)定位以切割或部分切割所述靶标。在一些实施方案中，所述功能结构域位于所述工程化的Cas12i效应蛋白的N末端。在一些实施方案中，所述功能结构域位于工程化的Cas12i效应蛋白的C末端。在一些实施方案中，所述工程化的Cas12i效应蛋白在N末端包含第一功能结构域，并在C末端包含第二功能结构域。在一些实施方案中，所述工程化Cas12i效应蛋白包含与一个或多个功能结构域融合的本文所述工程化的Cas12i核酸酶中任一种的催化失活突变体(dCas12i)。
在一些实施方案中，所述工程化的Cas12i效应蛋白是转录激活子。在一些实施方案中，所述工程化的Cas12i效应蛋白包含与反式激活结构域融合的本文所述工程化的Cas12i核酸酶中任一种的酶失活变体。在一些实施方案中，所述反式激活域选自下组：VP64、p65、HSF1、VP16、MyoD1、HSF1、RTA、SET7/9及其组合。在一些实施方案中，所述反式激活结构域包含VP64、p65和HSF1。在一些实施方案中，所述工程化的Cas12i效应蛋白包含两个分割型Cas12i效应多肽，每个都与反式激活结构域融合。
在一些实施方案中，所述工程化的Cas12i效应蛋白是转录阻遏物。在一些实施方案中，所述工程化的Cas12i效应蛋白包含与转录阻遏结构域融合的本文所述工程化的Cas12i核酸酶中任一种的酶失活变体。在一些实施方案中，所述转录阻遏物结构域选自下组：Krüppe1相关盒(KRAB)、EnR、NuE、NcoR、SID、SID4X及其组合。在一些实施方案中，所述工程化的Cas12i效应蛋白包含两个分割型Cas12i效应多肽，每个都与转录阻遏结构域融合。
在一些实施方案中，所述工程化的Cas12i效应蛋白是碱基编辑器，如胞嘧啶编辑器或腺苷编辑器。在一些实施方案中，所述工程化的Cas12i效应蛋白包含与核碱基编辑结构域融合的本文所述任何工程化的Cas12i核酸酶中任一种的酶失活变体，所述核碱基编辑结构域诸如为胞嘧啶碱基编辑(CBE)结构域或腺苷碱基编辑(ABE)域。在一些实施方案中，所述核碱基编辑结构域是DNA编辑结构域。在一些实施方案中，所述核碱基编辑结构域具有脱氨酶活性。在一些实施方案中，所述核碱基编辑结构域是胞嘧啶脱氨酶结构域。在一些实施方案中，所述核碱基编辑结构域是腺苷脱氨酶结构域。基于Cas核酸酶的示例性碱基编辑器描述于例如，WO2018/165629A1和WO2019/226953A1，其通过整体引用而并入本文。示例性CBE结构域包括但不限于：活化诱导的胞苷脱氨酶或AID(例如hAID)、载脂蛋白B mRNA编辑复合物或APOBEC(例如大鼠APOBEC1、hAPOBEC3A/B/C/D/E/F/G)和PmCDA1。示例性ABE结构域包括但不限于：TadA、 ABE8及其变体(参见例如Gaudelli et al.，2017，Nature 551：464-471；and Richter et al.，2020，Nature Biotechnology 38：883-891)。在一些实施方案中，所述功能性结构域是APOBEC1结构域，例如大鼠APOBEC1结构域。在一些实施方案中，所述功能性结构域是TadA结构域，例如大肠杆菌(E.coli)TadA结构域。在一些实施方案中所述工程化的Cas12i效应蛋白还包含一个或多个核定位序列。
腺苷脱氨酶
如本文所用，术语“腺苷脱氨酶”或“腺苷脱氨酶蛋白”是指蛋白质，多肽，或蛋白质或多肽的一个或多个功能结构域，其能够催化将腺嘌呤(或分子的腺嘌呤部分)转化为次黄嘌呤(或分子的次黄嘌呤部分)的水解脱氨反应，如下所示。在一些实施方式中，含腺嘌呤的分子是腺苷(A)，并且含次黄嘌呤的分子是肌苷(I)。含腺嘌呤的分子可以是脱氧核糖核酸(DNA)或核糖核酸(RNA)。
可与本发明工程化的Cas12i核酸酶中任一种的酶失活变体结合使用的腺苷脱氨酶包括但不限于称为作用于RNA的腺苷脱氨酶的酶家族成员(ADAR)，称为作用于tRNA的腺苷脱氨酶的酶家族成员(ADAT)，以及其他含腺苷脱氨酶结构域(ADAD)的家族成员。腺苷脱氨酶能够靶向RNA/DNA和RNA双链体中的腺嘌呤。实际上，Zheng等人(Nucleic Acids Res.2017，45(6)：3369-3377)证实ADAR可对RNA/DNA和RNA/RNA双链体进行腺苷至肌苷的编辑反应。在一些实施方式中，腺苷脱氨酶可以被修饰以增加其编辑RNA双链体的RNA/DNA异源双链体中的DNA的能力。
在一些实施方式中，腺苷脱氨酶源自一种或多种后生动物物种，包括但不限于哺乳动物、鸟类、青蛙、鱿鱼、鱼、蝇和蠕虫。在一些实施方式中，腺苷脱氨酶是人类、鱿鱼或果蝇腺苷脱氨酶。
在一些实施方式中，腺苷脱氨酶是人类ADAR，包括hADAR1、hADAR2、hADAR3。在一些实施方式中，腺苷脱氨酶是秀丽隐杆线虫(Caenorhabditis elegans)ADAR蛋白，包括ADR-1和ADR-2。在一些实施方式中，腺苷脱氨酶是果蝇ADAR蛋白，包括dAdar。在一些实施方式中，腺苷脱氨酶是鱿鱼(长翼鱿鱼(Loligo pealeii))ADAR蛋白，包括sqADAR2a和sqADAR2b。在一些实施方式中，腺苷脱氨酶是人类ADAT蛋白。在一些实施方式中，腺苷脱氨酶是果蝇ADAT蛋白。在一些实施方式中，腺苷脱氨酶是人类ADAD蛋白，包括TENR(hADAD1)和TENRL(hADAD2)。在一些实施方式中，腺苷脱氨酶是TadA8e。
在一些实施方式中，腺苷脱氨酶是TadA蛋白，例如大肠杆菌TadA。参见Kim等人，Biochemistry 45：6407-6416(2006)；Wolf等人，EMBO J.21：3841-3851(2002)。在一些实 施方式中，腺苷脱氨酶是小鼠ADA。参见Grunebaum等人，Curr.Opin.Allergy Clin.Immunol.13：630-638(2013)。在一些实施方式中，腺苷脱氨酶是人类ADAT2。参见Fukui等人，J.Nucleic Acids 2010：260512(2010)。在一些实施方式中，脱氨酶(例如腺苷或胞苷脱氨酶)是以下文献中描述的那些中的一种或多种：Cox等人，Science.2017年11月24日；358(6366)：1019-1027；Komore等人，Nature.2016年5月19日；533(7603)：420-4；以及Gaudelli等人，Nature.2017年11月23日；551(7681)：464-471。
在一些实施方式中，腺苷脱氨酶蛋白包含一个或多个脱氨酶结构域。不希望受特定理论的束缚，预期脱氨酶结构域用于识别双链核酸底物中所含的一个或多个靶腺苷(A)残基并将其转化为肌苷(I)残基。
胞苷脱氨酶
在一些实施方式中，脱氨酶是胞苷脱氨酶。如本文所用，术语“胞苷脱氨酶”或“胞苷脱氨酶蛋白”是指蛋白质、多肽或者蛋白质或多肽的一个或多个功能结构域，其能够催化将胞嘧啶(或分子的胞嘧啶部分)转化为尿嘧啶(或分子的尿嘧啶部分)的水解脱氨基反应。在一些实施方式中，含胞嘧啶的分子是胞苷(C)，并且含尿嘧啶的分子是尿苷(U)。所述含胞嘧啶的分子可以是脱氧核糖核酸(DNA)或核糖核酸(RNA)。
可与本发明工程化的Cas12i核酸酶中任一种的酶失活变体结合使用的胞苷脱氨酶包括但不限于被称为载脂蛋白B mRNA编辑复合物(APOBEC)家族脱氨酶的酶家族的成员，激活诱导的脱氨酶(AID)，或胞苷脱氨酶1(CDA1)。在一些实施方式中，APOBEC1脱氨酶、APOBEC2脱氨酶、APOBEC3A脱氨酶、APOBEC3B脱氨酶、APOBEC3C脱氨酶和APOBEC3D脱氨酶、APOBEC3E脱氨酶、APOBEC3F脱氨酶、APOBEC3G脱氨酶、APOBEC3H脱氨酶或APOBEC4脱氨酶中的脱氨酶。
在一些实施方式中，胞苷脱氨酶能够靶向DNA单链中的胞嘧啶。在一些实施方式中，胞苷脱氨酶可在存在于结合组分外部的单链上进行编辑。在一些实施方式中，胞苷脱氨酶可在局部化泡，例如由靶标编辑位点处但指导序列错配形成的局部化泡处编辑。在一些实施方式中，胞苷脱氨酶可包含有助于聚焦活性的突变，例如Kim等人，Nature Biotechnology(2017)35(4)：371-377(doi：10.1038/nbt.3803中所述的那些。
在一些实施方式中，胞苷脱氨酶源自一种或多种后生动物物种，包括但不限于哺乳动物、鸟类、青蛙、鱿鱼、鱼、蝇和蠕虫。在一些实施方式中，胞苷脱氨酶是人类、灵长类、牛、狗、大鼠或小鼠胞苷脱氨酶。
在一些实施方式中，胞苷脱氨酶是人类APOBEC，包括hAPOBEC1或hAPOBEC3。在一些实施方式中，胞苷脱氨酶是人类AID。
在一些实施方式中，胞苷脱氨酶蛋白包含一个或多个脱氨酶结构域。不希望受理论的束缚，预期脱氨酶结构域用于识别RNA双链体的单链泡中所含的一个或多个靶胞嘧啶(C)残基并将其转化为尿嘧啶(U)残基。
在一些实施方案中，所述工程化的Cas12i效应蛋白是主编辑器。基于Cas9的主编辑器描述于例如，A.Anzalone et al.，Nature，2019，576(7785)：149-157，其通过整体引用而并入本文。在一些实施方案中，所述工程化的Cas12i效应蛋白包含与逆转录酶结构域融合的本文所述工程化的Cas12i核酸酶中任一种的切口酶变体。在一些实施方案中，所述功能结构域是逆转录酶结构域。在一些实施方案中，所述逆转录酶结构域是M-MLV逆转录酶或其变体，例如具有D200N、T306K、W313F、T330P和L603W的一个或多个突变的M-MLV逆转录酶。在一些实施方案中，提供了包含所述主编辑器的工程化CRISPR/Cas12i系统。在一些实施方案中，所述工程化的CRISPR/Cas12i系统还包含第二Cas12i切口酶，例如基于与主编辑器的相同的工程化的Cas12i核酸酶。在一些实施方案中，所述工程化的CRISPR/Cas12i系统包含引导编辑指导RNA(pegRNA)，其包含引物结合位点和逆转录酶(RT)模板序列。
在一些实施方案中，本申请提供了具有与分割型Cas12i效应蛋白部分之一或两者缔合(即，结合或融合)的一个或多个(例如，1、2、3、4、5、6个或更多个)功能结构域的分割型Cas12i效应系统。所述功能结构域可以作为所第一和/或第二分割型Cas12i效应蛋白的一部分提供，作为该构建体内的融合物。所述功能结构域通常通过肽接头(诸如GS接头)与分割型Cas12i效应蛋白中其他部分融合(例如，分割型Cas12i效应蛋白部分)。这些功能结构域可用于基于催化失活Cas12i效应蛋白重新改换该分割型Cas12i效应系统的功能。
在一些实施方案中，所述工程化的Cas12i效应蛋白包含一个或多个核定位序列(NLS)和/或一个或多个核输出序列(NES)。示例性的NLS序列包括例如PKKKRKVPG(SEQ ID NO：66)和ASPKKKRKV(SEQ ID NO：67)。NLS和/或NES可以可操作地连接至所述工程化的Cas12i效应蛋白的N末端和/或C末端或所述工程化的Cas12i效应蛋白中的多肽链。在一些实施方式中，NLS和/或NES可以连接至本发明所述任何工程化的Cas12i核酸酶或其功能变体的N末端和/或C末端。
在一些实施方案中，所述工程化的Cas12i效应蛋白可以编码另外的组分，例如报告蛋白。在一些实施方案中，所述工程化的Cas12i效应蛋白包含荧光蛋白，例如GFP。这样的系统可以允许对基因组位点进行成像(例如，参见“Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System”Chen B et al.Cell 2013)。在一 些实施方案中所述工程化的Cas12i效应蛋白是可用于对基因组位点成像的可诱导分割型Cas12i效应系统。
在一些实施方案中，提供了一种工程化的Cas12i效应蛋白，其中所述效应蛋白能够诱导DNA分子中的双链断裂或单链断裂。
在一些实施方案中，提供了一种工程化的Cas12i效应蛋白，其中所述工程化的Cas12i核酸酶的功能衍生物为酶失活突变体，例如含有D599A、E833A、S883A、H884A、D886A、R900A和/或D1019A的Cas12i2核酸酶失活突变体(所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义)，和含有D647A、E894A和/或D948A的Cas12i1核酸酶失活突变体(所述氨基酸位置如SEQ ID NO：13所示的相应氨基酸位置所定义)。已知的Cas12i2核酸酶的酶失活突变体，如在US10808245B2和Huang X.et al.，Nature Communications，11，Article number：5241(2020)中描述的Cas12i2核酸酶的任何酶失活突变都可以和本申请中的突变组合在一起以提供工程化的Cas12i核酸酶的功能衍生物及其相应的效应蛋白。
工程化的CRISPR-Cas12i系统
在一些实施方式中，提供了一种工程化的CRISPR-Cas12i系统，包括：(a)本申请所述的任一种工程化的Cas12i效应蛋白(如工程化Cas12i核酸酶或其功能变体，例如SEQ ID NO：8所示CasXX)；以及(b)包含与靶序列互补的指导序列的指导RNA，或编码所述指导RNA的一种或多种核酸；
其中所述工程化的Cas12i效应蛋白和所述指导RNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰(如双链或单链切割、碱基编辑等)。在本说明书的上下文中，术语“修饰”涵盖核酸酶对于核酸双链或单链上的靶位点处的切割、碱基编辑、替换、修复等。
在一些实施方式中，所述工程化的CRISPR-Cas12i系统包括：(a)本文所述工程化的Cas12i效应蛋白中任一种(例如，任一所述工程化的Cas12i核酸酶或其功能变体，或基于该工程化的Cas12i核酸酶或其功能变体的切口酶、分割型Cas12i、转录阻遏物、转录激活子、碱基编辑器或主编辑器)；以及(b)包含与靶序列互补的指导序列的指导RNA，或编码所述指导RNA的一种或多种核酸；其中所述工程化的Cas12i效应蛋白和所述指导RNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合于包含所述靶序列的靶核酸并诱导所述靶核酸的修饰(如双链或单链切割、碱基编辑等)。在一些实施方案中，所述工程化的CRISPR-Cas12i系统包含编码所述工程化的Cas12i效应蛋白(如工程化Cas12i 核酸酶或其功能变体，例如SEQ ID NO：8所示CasXX)和/或所述指导RNA的一种或多种核酸。在一些实施方案中，所述工程化的CRISPR-Cas12i系统包含前体指导RNA阵列，其可以例如通过所述工程化的Cas12i效应蛋白加工成多个crRNA。在一些实施方案中，所述工程化的CRISPR-Cas12i系统包含一种或多种编码所述工程化的Cas12i效应蛋白和/或所述指导RNA的载体。在一些实施方案中，所述工程化的CRISPR-Cas12i系统包含核糖核蛋白(RNP)复合物，其包含结合至所述指导RNA的所述工程化的Cas12i效应蛋白。
本申请的工程化的CRISPR-Cas12i系统可包含任何合适的指导RNA。指导RNA(gRNA)可以包含能够与目标靶核酸(诸如细胞中的目标基因组位点)中的靶序列杂交的指导序列。在一些实施方案中，所述gRNA包含含有所述指导序列的CRISPRRNA(crRNA)序列。
通常，本文所述的crRNA包括直接重复序列和间隔区序列。在某些实施方案中，所述crRNA包括与指导序列或间隔区序列连接的直接重复序列、基本由或由它组成。在一些实施方案中，所述crRNA包括直接重复序列、间隔区序列和直接重复序列(DR-间隔区序列-DR)，其是前体crRNA(pre-crRNA)构型的典型特征。在一些实施方案中，所述crRNA包括截短的直接重复序列和间隔区序列，其是经加工的或成熟的crRNA的典型特征。在一些实施方案中，所述CRISPR-Cas12i效应蛋白与RNA指导序列形成复合物，并且所述间隔区序列将所述复合物引导至与靶核酸进行序列特异性结合，所述靶核酸与间隔区序列互补(如至少70％互补)。
在一些实施方案中，所述指导RNA是包含指导序列的crRNA。在一些实施方案中，所述工程化的CRISPR-Cas12i系统包含编码多个crRNA的前体指导RNA阵列。在一些实施方案中，所述Cas12i效应蛋白切割所述前体指导RNA阵列以产生多个crRNA。在一些实施方案中，所述工程化的CRISPR-Cas12i系统包含编码多个crRNA的前体指导RNA阵列，其中每个crRNA包含不同的指导序列。
构建体和载体
本文还提供了编码本文所述工程化的Cas12i效应蛋白(例如工程化Cas12i核酸酶或其功能变体)中任一种的构建体、载体和表达系统。在一些实施方案中，所述构建体、载体或表达系统还包含一种或多种gRNA或crRNA阵列。
“载体”是包含分离的核酸并且可以用于将所述分离的核酸递送至细胞内部的物质组合物。许多载体是本领域已知的，包括但不限于：线性多核苷酸、与离子或两亲性化合物缔合的多核苷酸、质粒和病毒。通常，合适的载体包含在至少一种生物中起作用的复制起点、启动子序列、方便的限制性核酸内切酶位点和一种或多种选择性标记物。术语 “载体”也应被解释为包括非质粒和非病毒化合物，其促进核酸转移到细胞中，诸如例如，聚赖氨酸化合物、脂质体等。
在一些实施方案中，所述载体是病毒载体。病毒载体的实例包括但不限于：腺病毒载体、腺相关病毒载体、慢病毒载体、逆转录病毒载体、牛痘载体、单纯疱疹病毒载体及其衍生物。在一些实施方案中，所述载体是噬菌体载体。病毒载体技术是本领域众所周知的，并且例如描述于Sambrook等人(2001，Molecular Cloning：A Laboratory Manual，Cold Spring Harbor Laboratory，New York)，以及其他病毒学和分子生物学手册。
已经开发了许多基于病毒的系统，用于将基因转移到哺乳动物细胞中。例如，逆转录病毒为基因递送系统提供了便利的平台。可以使用本领域已知的技术将异源核酸插入载体，并包装在逆转录病毒颗粒中。然后可以分离重组病毒，并在体外或离体递送至所述工程化的哺乳动物细胞。许多逆转录病毒系统是本领域已知的。在一些实施方案中，使用腺病毒载体。许多腺病毒载体是本领域已知的。在一些实施方案中，使用慢病毒载体。在一些实施方案中，使用自灭活的慢病毒载体。
在某些实施方案中，所述载体是腺相关病毒(AAV)载体，例如AAV2、AAV8或AAV9，其可以按包含至少1×10 5个颗粒(也称为颗粒单位，pu)的单次给药施用腺病毒或腺相关病毒。在一些实施方案中，所述给药量是至少约1×10 6个颗粒，至少约1×10 7个颗粒，至少约1×10 8个颗粒，或至少约1×10 9个颗粒的腺相关病毒。递送方法和给药量描述于例如WO 2016205764和美国专利No.8,454,972，其通过整体引用而并入本文。
在一些实施方案中，所述载体是重组腺相关病毒(rAAV)载体。例如，在一些实施方案中，可以使用经修饰的AAV载体递送。经修饰的AAV载体可以基于几种衣壳类型中的一种或多种，包括AAV1、AV2、AAV5、AAV6、AAV8、AAV8.2、AAV9、AAV rh10，经修饰的AAV载体(例如经修饰的AAV2，经修饰的AAV3，经修饰的AAV6)和假型AAV(例如AAV2/8，AAV2/5和AAV2/6)。可用于产生rAAV颗粒的示例性AAV载体和技术是本领域已知的(参见例如Aponte-Ubillus et al.(2018)Appl.Microbiol.Biotechnol.102(3)：1045-54；Zhong et al.(2012)J.Genet.Syndr.Gene Ther.S1：008；West et al.(1987)Virology 160：38-47(1987)；Tratschin et al.(1985)Mol.Cell.Biol.5：3251-60；美国专利Nos.4,797,368和5,173,414；国际公开第WO2015/054653号和第WO93/24641号，其各自通过引用并入本文)。
用于递送Cas9和其他Cas蛋白的任何已知的AAV载体都可以用于递送本申请的工程化的Cas12i系统。
在一些实施方案中，rAAV构建体可经肠内向受试者施用。在一些实施方案中， rAAV构建体可经肠胃外向受试者施用。在一些实施方案中，rAAV颗粒可经皮下、眼内、玻璃体内、视网膜下、静脉内(IV)、脑室内、肌内、鞘内(IT)、脑池内、腹膜内、经由吸入、局部或通过直接注射到一种或多种细胞、组织或器官。在一些实施方案中，rAAV颗粒可通过注射到肝动脉或门静脉中向受试者施用。
将载体引入哺乳动物细胞的方法是本领域已知的。可以通过物理、化学或生物学方法将载体转移到宿主细胞中。
用于将载体引入宿主细胞的物理方法包括：磷酸钙沉淀、脂质转染、粒子轰击、显微注射、电穿孔等。产生包含载体和/或外源核酸的细胞的方法是本领域众所周知的。参见，例如Sambrook et al.(2001)Molecular Cloning：A Laboratory Manual，Cold Spring Harbor Laboratory，New York。在一些实施方案中，通过电穿孔将所述载体引入所述细胞。
用于将异源核酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体已成为将基因插入哺乳动物例如人细胞的最广泛使用的方法。
用于将载体引入宿主细胞的化学方法包括胶体分散系统如大分子复合物、纳米胶囊、微球、珠子和基于脂质的系统，所述基于脂质的系统包括水包油乳液、胶束、混合胶束和脂质体。用作体外递送载体的示例性胶体系统是脂质体(例如，人工膜囊泡)。在一些实施方案中，所述工程化的CRISPR-Cas12i系统以RNP的形式在纳米颗粒中递送。
在一些实施方案中，编码所述CRISPR-Cas12i系统或其组分的载体或表达系统包含一种或多种可选择或可检测的标记物，该标记物提供了分离或有效选择含有和/或已被CRISPR-Cas12i系统(例如在早期和大规模)修饰的细胞的手段。
报告基因可用于鉴定潜在转染的细胞和评估调节序列的功能。通常，报告基因是在受体生物体或组织中不存在或不表达的基因，其编码的多肽的表达是通过某些易于检测的性质(例如酶活性)来证明。在将DNA引入受体细胞后的合适时间测定报告基因的表达。合适的报告基因可以包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌的碱性磷酸酶的基因或绿色荧光蛋白基因(例如Ui-Tei et al.FEBS Letters 479：79-82(2000))。
确认宿主细胞中异源核酸的存在的其他方法包括例如本领域技术人员众所周知的分子生物学测定，诸如Southern和Northern印迹、RT-PCR和PCR；生化测定，例如通过免疫学方法(如ELISA和Western印迹)检测特定肽的存在或不存在。
在一些实施方案中，编码所述工程化的Cas12i效应蛋白和/或所述指导RNA的核酸序列与启动子可操作地连接。在一些实施方案中，所述启动子是相对于使用所述工程化的CRISPR-Cas12i系统进行工程化改造的细胞的内源启动子。例如，可以使用本领域已知的任何方法将编码所述工程化的Cas12i效应蛋白的核酸敲入工程化的哺乳动物细胞的 基因组中位于内源性启动子下游处。在一些实施方案中，所述内源启动子是丰富蛋白(如β-肌动蛋白)的启动子。在一些实施方案中，所述内源启动子是可诱导的启动子，例如，可通过工程化的哺乳动物细胞的内源激活信号来诱导。在一些实施方案中，其中所述工程化的哺乳动物细胞是T细胞，所述启动子是T细胞活化依赖性启动子(诸如IL-2启动子、NFAT启动子或NFκB启动子)。
在一些实施方案中，所述启动子是相对于使用所述工程化的CRISPR-Cas12i系统进行工程化改造的细胞的异源启动子。已经探索了多种启动子以在哺乳动物细胞中表达基因，并且本领域中已知的任何启动子都可以用于本申请中。启动子可大致分为组成型启动子或受调节的启动子，例如诱导型启动子。
在一些实施方案中，编码所述工程化的Cas12i效应蛋白和/或所述指导RNA的核酸序列与组成型启动子可操作地连接。组成型启动子允许异源基因(也称为转基因)在宿主细胞中组成型表达。本文考虑的示例性组成型启动子包括但不限于：巨细胞病毒(CMV)启动子、人延伸因子-1α(hEF1α)、泛素C启动子(UbiC)、磷酸甘油激酶启动子(PGK)、猿猴病毒40早期启动子(SV40)以及鸡β-肌动蛋白启动子与CMV早期增强子(CAG)耦联。在一些实施方案中，所述启动子是CAG启动子，其包含巨细胞病毒(CMV)早期增强子元件、启动子、鸡β-肌动蛋白基因的第一外显子和第一内含子，以及兔β-珠蛋白基因的剪接受体。
在一些实施方案中，编码所述工程化的CRISPR-Cas12i效应蛋白和/或所述指导RNA的核酸序列可操作地连接至诱导型启动子。诱导型启动子属于受调控的启动子类型。所述诱导型启动子可以通过一种或多种条件如物理条件、微环境或宿主细胞的生理状态、诱导物(即，诱导剂)或其组合来诱导。在一些实施方案中，所述诱导条件选自下组：诱导剂、辐照(如电离辐射、光)、温度(如热)、氧化还原状态、肿瘤环境和待通过工程化的CRISPR-Cas12i系统进行工程化改造的细胞的活化状态。在一些实施方案中，所述启动子可被小分子诱导剂诸如化合物诱导。在一些实施方案中，所述小分子选自下组：强力霉素、四环素、醇、金属或类固醇。化学诱导的启动子已被最广泛地研究。这样的启动子包括其转录活性受小分子化学物质例如强力霉素、四环素、醇、类固醇、金属和其他化合物的存在或不存在调节的启动子。具有逆四环素受控反式激活因子(rtTA)和四环素响应元件启动子(TRE)的强力霉素诱导系统是目前最成熟的系统。WO9429442描述了通过四环素响应性启动子严格控制真核细胞中基因表达。WO9601313公开了四环素调节的转录调节剂。另外，诸如Tet-on系统之类的Tet技术已描述于例如TetSystems.com网站。在本申请中，任何已知的化学调节的启动子均可用于驱动编码所述工程化的CRISPR-Cas12i蛋白和/或所述指导RNA的表达。
在一些实施方案中，编码所述工程化的Cas12i效应蛋白的核酸序列是经过密码子优化的。
在一些实施方案中，提供了表达构建体，其包含经密码子优化的序列，编码所述工程化的Cas12i效应蛋白的该序列连接到BPK2104-ccdB载体上。在一些实施方案中，所述表达构建体编码与所述工程化的Cas12i效应蛋白的C末端可操作连接的标签(例如10×His标签)。
在一些实施方案中，每种工程化的分割型Cas12i构建体编码荧光蛋白诸如GFP或RFP。所述报告蛋白可以用于评估所述工程化的Cas12i蛋白的共定位和/或二聚化，例如使用显微镜。可以使用编码自切割肽诸如T2A、P2A、E2A或F2A肽的序列，将编码工程化的Cas12i效应蛋白的核酸序列与编码另外的组分的核酸序列融合。
在一些实施方案中，提供了用于哺乳动物细胞(例如人细胞)的表达构建体，所述构建体包含编码所述工程化的Cas12i效应蛋白的核酸序列。在一些实施方案中，所述表达构建体包含经密码子优化的序列，所述序列编码插入pCAG-2A-eGFP载体中的所述工程化的Cas12i效应蛋白，从而使Cas12i蛋白可操作地连接至eGFP。在一些实施方案中，提供第二载体用于在哺乳动物细胞(例如人细胞)中表达指导RNA(例如crRNA或前体crRNA阵列)。在一些实施方案中，编码所述指导RNA的序列在pUC19-U6-i2-cr RNA载体主链中表达。
在一些实施方式中，将CRISPR-Cas12i系统的一个或多个元件表达的一个或多个载体引入宿主细胞，使得CRISPR-Cas12i系统的元件的表达引导核酸靶向复合物在一个或多个靶位点的形成。例如，Cas12i核酸靶向效应酶和核酸靶向指导RNA可各自可操作地连接至分开的载体上的分开的调控元件。可将核酸靶向系统的RNA递送至转基因Cas12i核酸靶向效应蛋白动物或哺乳动物，例如，组成性或诱导性或条件性表达核酸靶向效应蛋白的动物或哺乳动物；或以其他方式表达核酸靶向效应蛋白或者具有含有核酸靶向效应蛋白的细胞的动物或哺乳动物，例如通过事先向其施用编码并表达体内核酸靶向效应蛋白的一个或多个载体。或者，可将由相同或不同调控元件表达的两个或更多个元件组合在单个载体中，而一个或多个额外载体提供不包含在第一载体中的核酸靶向系统的任何组分。组合在单个载体中的核酸靶向系统元件可以任何合适的方向排列，例如一个元件位于相对于第二元件的(“上游”)5′或相对于第二元件的(“下游”)3′。一个元件的编码序列可位于第二元件的编码序列的相同或相反链上，并以相同或相反的方向定向。在一些实施方式中，单个启动子驱动编码Cas12i核酸靶向效应蛋白和核酸靶向指导RNA的转录物的表达，所述转录物嵌入一个或多个内含子序列内(例如，各自在不同的内含子中， 两个或更多个在至少一个内含子中，或全部在单个内含子中)。在一些实施方式中，核酸靶向效应蛋白和核酸靶向指导RNA可以可操作地连接至同一启动子并从同一启动子表达。用于表达核酸靶向系统的一个或多个元件的递送媒介物、载体、粒子、纳米粒子、制剂及其组分如前述文件例如WO 2014/093622(PCT/US2013/074667)中所使用。在一些实施方式中，载体包含一个或多个插入位点，例如限制性核酸内切酶识别序列(也称为“克隆位点”)。在一些实施方式中，一个或多个插入位点(例如，约或大于约1、2、3、4、5、6、7、8、9、10个或更多个插入位点)位于一个或多个载体的一个或多个序列元件的上游和/或下游。当使用多个不同的指导序列时，单个表达构建体可用于将核酸靶向活性靶向细胞内的多个不同的相应靶序列。例如，单个载体可包含约或大于约1、2、3、4、5、6、7、8、9、10、15、20个或更多个指导序列。在一些实施方式中，可提供约或大于约1、2、3、4、5、6、7、8、9、10个或更多个这样的含指导序列的载体，并任选地递送至细胞。在一些实施方式中，载体包含与编码核酸靶向效应蛋白的酶编码序列可操作地连接的调控元件。Cas12i核酸靶向效应蛋白或者一种或多种核酸靶向指导RNA可分开递送；并且有利地，这些中的至少一种经由粒子复合物递送。可在核酸靶向指导RNA之前递送核酸靶向效应蛋白mRNA，以留出时间表达Cas12i核酸靶向效应蛋白。核酸靶向效应蛋白mRNA可在施用核酸靶向指导RNA之前1-12小时(优选约2-6小时)施用。或者，核酸靶向效应蛋白mRNA和核酸靶向指导RNA可一起施用。有利地，可在初次施用核酸靶向效应蛋白mRNA+指导RNA后1-12小时(优选约2-6小时)施用指导RNA的第二加强剂量。核酸靶向效应蛋白mRNA和/或指导RNA的其他施用可能对实现最有效的基因组修饰水平有用。
在一些实施方式中，提供了一种CRISPR-Cas12i系统，其包含：(1)任一所述的工程化的Cas12i效应蛋白(如任一所述工程化的Cas12i核酸酶或其功能变体)或编码任一所述工程化的Cas12i效应蛋白的多核苷酸；和(2)crRNA或编码所述crRNA的多核苷酸，所述crRNA包含：(i)能够与靶DNA的靶序列杂交的间隔序列，和(ii)连接至所述间隔序列的能够引导所述工程化的Cas12i效应蛋白结合至所述crRNA以形成靶向所述靶序列的CRISPR-Cas12i复合物的直接重复序列。
在一些实施方式中，提供了一种CRISPR-Cas12i系统，其包括一个或多个载体，所述一个或多个载体包含：(1)第一调控元件，所述第一调控元件可操作地连接至编码任一所述工程化的Cas12i效应蛋白(如任一所述工程化的Cas12i核酸酶或其功能变体)的核苷酸序列；和(2)第二调控元件，所述第二调控元件可操作地连接至编码crRNA的多核苷酸，所述crRNA包含：(i)能够与靶DNA的靶序列杂交的间隔序列，和(ii)连接至所述间隔序 列的能够引导所述工程化的Cas12i效应蛋白结合至所述crRNA以形成靶向所述靶序列的CRISPR-Cas12i复合物的直接重复序列；其中所述第一调控元件和所述第二调控元件位于所述CRISPR-Cas12i系统的相同或不同载体上。
在某些实施方式中，所述第一调控元件和所述第二调控元件位于所述CRISPR-Cas12i系统的不同载体上。在某些实施方式中，所述第一调控元件和所述第二调控元件位于所述CRISPR-Cas12i系统的相同载体上。在某些实施方式中，所述第一调控元件和所述编码工程化的Cas12i效应蛋白的核苷酸序列在所述第二调控元件和所述编码crRNA的多核苷酸的上游。在某些实施方式中，所述第一调控元件和所述编码工程化的Cas12i效应蛋白的核苷酸序列在所述第二调控元件和所述编码crRNA的多核苷酸的下游。在某些实施方式中，所述第一调控元件和所述第二调控元件相同。在某些实施方式中，所述第一调控元件和所述第二调控元件不同。
在一些实施方式中，提供了一种CRISPR-Cas12i系统，其包括一个载体，所述载体包含：(1)编码任一所述工程化的Cas12i效应蛋白(如任一所述工程化的Cas12i核酸酶或其功能变体)的多核苷酸；(2)编码crRNA的多核苷酸，所述crRNA包含：(i)能够与靶DNA的靶序列杂交的间隔序列，和(ii)连接至所述间隔序列的能够引导所述工程化的Cas12i效应蛋白结合至所述crRNA以形成靶向所述靶序列的CRISPR-Cas12i复合物的直接重复序列；和(3)调控元件，所述调控元件可操作地连接至编码所述工程化的Cas12i效应蛋白的多核苷酸和编码所述crRNA的多核苷酸。
在某些实施方式中，所述载体从5’到3’包含所述调控元件、编码所述工程化的Cas12i效应蛋白的多核苷酸和编码所述crRNA的多核苷酸。在某些实施方式中，所述载体从5’到3’包含所述调控元件、编码所述crRNA的多核苷酸和编码所述工程化的Cas12i效应蛋白的多核苷酸。在某些实施方式中，编码所述crRNA的多核苷酸和编码所述工程化的Cas12i效应蛋白的多核苷酸由连接序列连接，例如编码P2A、T2A、E2A、F2A、BmCPV 2A、BmIFV 2A、(GS)n(SEQ ID NO：74)、(GGGS)n(SEQ ID NO：75)和(GGGGS)n(SEQ ID NO：76)中任一项的多核苷酸序列(其中n是至少为1的整数)，或任一IRES、SV40、CMV、UBC、EF1α、PGK和CAGG的多核苷酸序列，或其任何组合。
在一些实施方式中，本发明所述CRISPR-Cas12i系统的组分可以各种形式递送，例如DNA/RNA或RNA/RNA或蛋白质RNA的组合。例如，工程化的Cas12i效应蛋白(如任一所述工程化的Cas12i核酸酶或其功能变体)可作为编码DNA的多核苷酸或编码RNA的多核苷酸或作为蛋白质被递送。所述指导物可作为DNA编码多核苷酸或RNA被递送。可以使用混合的递送形式。
在一些方面，本发明提供了包括将一个或多个多核苷酸例如如本文所述的一个或多个载体、其一个或多个转录物和/或从其转录的一个或多个蛋白质递送至宿主细胞的方法。
III.使用方法
本申请提供了使用本文所述工程化的Cas12i效应蛋白(如任一所述工程化的Cas12i核酸酶或其功能变体)或CRISPR-Cas12i系统中任一种在体外、离体或体内检测靶核酸或修饰核酸的方法，以及使用所述工程化的Cas12i效应蛋白或CRISPR-Cas12i系统进行治疗(如基因编辑)或诊断的方法。还提供了本文所述工程化的Cas12i效应蛋白或CRISPR-Cas12i系统用于检测或修饰细胞中的核酸，以及用于治疗或诊断受试者的疾病或病况的用途；以及包含所述工程化的Cas12i效应蛋白中任一种或所述工程化的CRISPR-Cas12i系统的一种或多种组分的组合物在制备用于检测或修饰细胞中的核酸以及用于治疗或诊断受试者的疾病或病况的药物中的用途。
检测样品中靶核酸的方法
本申请还提供了使用具有改善的活性的所述工程化的Cas12i效应蛋白或CRISPR-Cas12i系统中任一种来检测靶核酸的方法。使用Cas12i效应蛋白作为检测试剂可利用以下发现：一旦通过检测到靶DNA而被激活，V型CRISPR/Cas蛋白(例如，Cas12i)可混杂地切割非靶向单链DNA(ssDNA或RNA，即指导RNA的指导序列不与之杂交的单链核酸)。因此，当样品中存在靶DNA(双链或单链)时(例如，在某些情况下超过阈值量)，结果是样品中单链核酸的切割，这可使用任何方便的检测方法进行检测(例如，使用加标签的单链检测核酸如DNA或RNA)。Cas12i可以切割ssDNA和ssRNA。例如使用Cas蛋白作为检测试剂的方法描述于US10253365和WO2020/056924，其通过整体引用而并入本文。
在一些实施方案中，提供了检测样品中的靶DNA(例如，双链或单链)的方法，其包括：(a)使所述样品与以下项接触：(i)本文所述工程化的Cas12i效应蛋白(如任一所述工程化的Cas12i核酸酶或其功能变体)中任一种；(ii)指导RNA，其包含与所述靶DNA杂交的指导序列；和(iii)检测核酸，其为单链的(即“单链检测核酸”)并且不与所述指导RNA的指导序列杂交；以及(b)测量通过所述工程化的Cas12i效应蛋白切割所述单链检测核酸而产生的可检测信号。在某些情况下，所述单链检测核酸包括发射荧光的染料对(例如，发射荧光的染料对是荧光共振能量转移(FRET)对、猝灭剂/荧光对)。在某些情况下，所述靶DNA是病毒DNA(例如，乳头状病毒、肝DNA病毒、疱疹病毒、腺病毒、痘病毒、细小病毒等)。在一些实施方案中，所述单链检测核酸是DNA。在一些实施方案中，所 述单链检测核酸是RNA。
本公开的用于检测样品中的靶DNA(单链或双链)的方法，可以以高灵敏度检测靶DNA。在一些情况下，本公开的方法可以用于检测存在于包含多个DNA(包括所述靶DNA和多个非靶DNA)的样品中的靶DNA，其中所述靶DNA以每10 7个非靶DNA中的一个或更多个拷贝存在(例如，每10 6个非靶DNA中的一个或更多个拷贝，每10 5个非靶DNA中的一个或更多个拷贝，每10 4个非靶DNA中的一个或更多个拷贝，每10 3个非靶DNA中的一个或更多个拷贝，每10 2个非靶DNA中的一个或更多个拷贝，每50个非靶DNA中的一个或更多个拷贝，每20个非靶DNA中的一个或更多个拷贝，每10个非靶DNA中的一个或更多个拷贝或每5个非靶DNA中的一个或更多个拷贝)。在一些实施方案中，与所述参比Cas12i核酸酶相比，本文所述工程化的Cas12i效应蛋白可以以更高的灵敏度检测靶DNA。在一些实施方案中，与所述参比Cas12i核酸酶相比，所述工程化的Cas12i效应蛋白可以以10％、15％、20％、25％、30％、40％、50％、60％、70％、80％、90％、95％或更高的灵敏度检测靶DNA。
修饰的方法
在一些实施方案中，本申请提供了修饰包含靶序列的靶核酸的方法，所述方法包括使所述靶核酸与本文所述工程化的CRISPR-Cas12i系统中任一种接触。在一些实施方案中，所述方法在体外进行。在一些实施方案中，所述靶核酸存在于细胞中。在一些实施方案中，所述细胞是细菌细胞、酵母细胞、哺乳动物细胞、植物细胞或动物细胞。在一些实施方案中，所述方法离体进行。在一些实施方案中，所述方法在体内进行。靶核酸修饰包括但不限于靶核酸单链切割、双链切割、碱基替换、碱基插入、碱基删除、突变(如致病突变)序列修复等。
在一些实施方案中，通过所述工程化的CRISPR-Cas12i系统切割所述靶核酸或改变所述靶核酸中的靶序列。在一些实施方案中，通过所述工程化的CRISPR-Cas12i系统改变所述靶核酸的表达。在一些实施方案中，所述靶核酸是基因组DNA。在一些实施方案中，所述靶序列与疾病或病况相关，例如基于靶序列的错误表达(如过表达、不表达、或表达致病RNA或蛋白)。在一些实施方案中，所述工程化的CRISPR-Cas12i系统包含编码多个crRNA的前体指导RNA阵列，其中每个crRNA包含不同的指导序列。
在一些实施方案中，本申请提供了处理与个体细胞中的靶核酸相关的疾病或病况的方法，其包括使用本文所述方法中任一种来修饰所述个体细胞中的所述靶核酸，从而处理所述疾病或病况。在一些实施方案中，所述疾病或病况选自下组：癌症、心血管疾病、遗传性疾病(例如基因缺陷性疾病，如镰刀型红细胞贫血(SCD)或β-地中海贫血(TDT))、 自身免疫疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。
本文所述工程化的CRISPR-Cas12i系统可以用多种方式修饰细胞中的靶核酸，这取决于所述CRISPR-Cas12i系统中工程化的Cas12i效应蛋白的类型。在一些实施方案中，所述方法诱导靶核酸中的位点特异性切割。在一些实施方案中，所述方法在细胞如细菌细胞、植物细胞或动物细胞(例如哺乳动物细胞)中切割基因组DNA。在一些实施方案中，所述方法通过切割细胞中的基因组DNA来杀死细胞。在一些实施方案中所述方法在细胞中切割病毒核酸。
在一些实施方案中，所述方法改变(如增加或减少)细胞中所述靶核酸的表达水平。在一些实施方案中，所述方法例如基于融合至反式激活结构域的无酶活性的Cas12i蛋白，使用工程化的Cas12i效应蛋白来提高细胞中所述靶核酸的表达水平。在一些实施方案中，所述方法例如基于融合至转录阻遏结构域的无酶活性的Cas12i蛋白，使用工程化的Cas12i效应蛋白来降低细胞中所述靶核酸的表达水平。在一些实施方案中，所述方法例如基于融合至表观遗传修饰结构域的无酶活性的Cas12i蛋白，使用工程化的Cas12i效应蛋白将表观遗传修饰引入细胞中的所述靶核酸或靶核酸相关蛋白(如结合至靶核酸或其临近的蛋白，例如转录因子或组蛋白)。本文所述工程化的Cas12i系统可用于将其他修饰引入所述靶核酸，这取决于所述工程化的Cas12i效应蛋白所包含的功能结构域。
在一些实施方案中，所述方法改变细胞中所述靶核酸中的靶序列。在一些实施方案中，所述方法将突变引入细胞中的所述靶核酸，比如将致病突变突变为非致病序列(例如，使用与包含腺苷脱氨酶或胞苷脱氨酶融合的任一本发明所述dCas12i)。在一些实施方案中，所述方法使用一种或多种内源性DNA修复途径，如非同源末端连接(NHEJ)或同源定向重组(HDR)，在细胞中修复靶DNA中诱导的双链断裂，作为由CRISPR复合物进行序列特异性切割的结果。示例性的突变包括但不限于：插入、缺失、替换和移码。在一些实施方案中，使用pegRNA来同时进行靶序列切割和提供DNA修复模板。在一些实施方案中，所述方法在靶位点处插入供体DNA。在一些实施方案中，供体DNA的插入导致将选择性标记物或报告蛋白引入所述细胞。在一些实施方案中，供体DNA的插入导致基因的敲入。在一些实施方案中，供体DNA的插入导致敲除突变。在一些实施方案中，供体DNA的插入导致替换突变诸如单核苷酸替换。在一些实施方案中，所述方法诱导细胞的表型变化。
在一些实施方案中，所述工程化的CRISPR-Cas12i系统用于基因电路(genetic circuit)的一部分，或用于将基因电路插入细胞的基因组DNA中。本文所述的诱导剂控制的工程化的分割型Cas12i效应蛋白尤其可用作基因电路的组分。基因电路可用于基因疗法。设 计和使用基因电路的方法和技术是本领域已知的。可以进一步参考例如Brophy，Jennifer AN，and Christopher A.Voigt.″Principles of genetic circuit design.″Nature methods 11.5(2014)：508。
本文所述工程化的CRISPR-Cas12i系统可用于修饰多种靶核酸。在一些实施方案中，所述靶核酸在细胞中。在一些实施方案中，所述靶核酸是基因组DNA。在一些实施方案中，所述靶核酸是染色体外DNA。在一些实施方案中，所述靶核酸对于细胞是外源的。在一些实施方案中，所述靶核酸是病毒核酸诸如病毒DNA。在一些实施方案中，所述靶核酸是细胞中的质粒。在一些实施方案中，所述靶核酸是经水平转移(horizontally transferred)的质粒。在一些实施方案中，所述靶核酸是RNA。
在一些实施方案中，所述靶核酸是经分离的核酸诸如经分离的DNA。在一些实施方案中，所述靶核酸存在于无细胞的环境中。在一些实施方案中，所述靶核酸是经分离的载体诸如质粒。在一些实施方案中，所述靶核酸是经分离的线性DNA片段。
本文描述的方法适用于任何合适的细胞类型。在一些实施方案中，所述细胞是细菌、酵母细胞、真菌细胞、藻类细胞、植物细胞或动物细胞(例如，哺乳动物细胞，如人细胞)。在一些实施方案中，所述细胞是自然来源的诸如由组织活检分离出的细胞。在一些实施方案中，所述细胞是从体外培养的细胞系分离的细胞。在一些实施方案中，所述细胞来自原代细胞系。在一些实施方案中，所述细胞来自永生化细胞系。在一些实施方案中，所述细胞是基因工程化的细胞。
在一些实施方案中，所述细胞是选自下组的生物的动物细胞：牛、绵羊、山羊、马、猪、鹿、鸡、鸭、鹅、兔和鱼。
在一些实施方案中，所述细胞是选自下组的生物的植物细胞：玉米、小麦、大麦、燕麦、水稻、大豆、油棕、红花、芝麻、烟草、亚麻、棉花、向日葵、珍珠小米、粟米、高粱、油菜、大麻、蔬菜作物、饲料作物、工业作物、木本作物和生物质作物。
在一些实施方案中，所述细胞是哺乳动物细胞。在一些实施方案中，所述细胞是人细胞。在一些实施方案中，所述人细胞是人胚胎肾293T(HEK293T或293T)细胞或HeLa细胞。在一些实施方案中，所述细胞是人胚肾(HEK293T)细胞。在一些实施方案中，所述细胞是小鼠Hepa1-6细胞。在一些实施方案中，所述哺乳动物细胞选自下组：免疫细胞、肝细胞、肿瘤细胞、干细胞、血液细胞、神经细胞、合子、肌肉细胞(如心肌细胞)和皮肤细胞。
在一些实施方案中，所述细胞是选自下组的免疫细胞：细胞毒性T细胞、辅助T细胞、天然杀伤(NK)T细胞、iNK-T细胞、NK-T样细胞、γδT细胞、肿瘤浸润性T细胞和 树突状细胞(DC)激活的T细胞。在一些实施方案中，所述方法产生经修饰的免疫细胞，诸如CAR-T细胞或TCR-T细胞。
在一些实施方案中，所述细胞是胚胎干(ES)细胞、诱导性多能干(iPS)细胞、配子的祖细胞、配子、合子或胚胎中的细胞。
本文所述的方法可以用于在体内、离体或体外修饰靶细胞，并且可以用改变所述细胞的方式进行，以使得一旦被修饰，所述经修饰的细胞的后代或细胞系就保留经改变的表型。所述经修饰的细胞和后代可以是多细胞生物的一部分，诸如具有离体或体内应用(如基因组编辑和基因疗法)的植物或动物。
在一些实施方案中，所述方法离体进行。在一些实施方案中，将所述工程化的CRISPR-Cas12i系统引入细胞后，所述经修饰的细胞(例如，哺乳动物细胞)离体繁殖。在一些实施方案中，将所述经修饰的细胞培养以繁殖至少约1天、2天、3天、4天、5天、6天、7天、10天、12天或14天中任一个。在一些实施方案中，将所述经修饰的细胞培养不超过约1天、2天、3天、4天、5天、6天、7天、10天、12天或14天中任一个。在一些实施方案中，进一步评价或筛选所述经修饰的细胞以选择具有一种或多种所需表型或特性的细胞。
在一些实施方案中，所述靶序列是与疾病或病况相关的序列。示例性的疾病或病况包括但不限于：癌症、心血管疾病、遗传性疾病、自身免疫性疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。在一些实施方案中，所述疾病或病况是遗传疾病。在一些实施方案中，所述疾病或病况是单基因疾病或病况。在一些实施方案中，所述疾病或病况是多基因疾病或病况。
在一些实施方案中，与野生型序列相比，所述靶序列具有突变。在一些实施方案中，所述靶序列具有与疾病或病况相关的单核苷酸多态性(SNP)。
在一些实施方案中，插入所述靶核酸中的供体DNA编码选自下组的生物产物：报告蛋白、抗原特异性受体、治疗性蛋白、抗生素抗性蛋白、RNAi分子、细胞因子、激酶、抗原、抗原特异性受体、细胞因子受体和自杀多肽。在一些实施方案中，所述供体DNA编码治疗性蛋白。在一些实施方案中，所述供体DNA编码可用于基因疗法的治疗性蛋白。在一些实施方案中，所述供体DNA编码治疗性抗体。在一些实施方案中，所述供体DNA编码工程化的受体，诸如嵌合抗原受体(CAR)或工程化的TCR。在一些实施方案中，所述供体DNA编码治疗性RNA，诸如小RNA(例如，siRNA、shRNA或miRNA)或长的非编码RNA(lincRNA)。
本文所述的方法可以用于在两个或更多个(例如2、3、4、5、6、8、10个或更多)不 同的靶位点处进行多重基因编辑或调节。在一些实施方案中，所述方法检测或修饰多个靶核酸或靶核酸序列。在一些实施方案中，所述方法包括使靶核酸与包含多个(例如2、3、4、5、6、8、10个或更多)crRNA序列的指导RNA接触，其中每个crRNA均包含不同的靶序列。
还提供了包含经修饰的靶核酸的工程化的细胞，所述细胞使用本文所述方法中任一种来产生。所述工程化的细胞可以用于细胞疗法。可以使用本文所述用于细胞疗法的方法将自体或同种异体细胞用于制备工程化的细胞。
本文所述的方法还可用于生成细胞(例如哺乳动物细胞)的等基因系以研究遗传变体。
还提供了包含本文所述工程化的细胞的工程化非人类动物。在一些实施方案中，所述工程化的非人类动物是经过基因组编辑的非人类动物。所述工程化的非人类动物可以用作疾病模型。
产生非人类基因组编辑或转基因动物的技术是本领域众所周知的，包括但不限于：原核显微注射、病毒感染、对胚胎干细胞和诱导性多能干(iPS)细胞的转化。可以使用的详细方法包括但不限于Sundberg和Ichiki描述的方法(2006，Genetically Engineered Mice Handbook，CRC Press)和Gibson描述的方法(2004，A Primer Of Genome Science 2nd ed.Sunderland，Mass.：Sinauer)。
所述工程化的动物可以是任何合适的物种，包括但不限于：牛、马、羊、犬、鹿、猫科动物，山羊、猪、灵长类，以及了解较少的哺乳动物诸如大象、鹿、斑马或骆驼。
治疗方法
在一些实施方式中，提供了前述工程化的CRISPR-Cas12i系统在制备治疗与个体的细胞中靶核酸相关的疾病或病症的药物中的用途。在一些实施方式中，提供了前述工程化的CRISPR-Cas12i系统在治疗与个体的细胞中靶核酸相关的疾病或病症的方法。
在一些实施方式中，本发明提供了一种治疗有需要的受试者(如人)的疾病的方法，所述方法包括向所述受试者施用(如静脉注射或滴注)本发明的CRISPR-Cas12i系统，其包含：(1)任一所述工程化的Cas12i效应蛋白(如任一所述工程化的Cas12i核酸酶或其功能变体)，或编码所述工程化的Cas12i效应蛋白的多核苷酸；和(2)crRNA，或编码所述crRNA的多核苷酸，所述crRNA包含：(i)能够和与该疾病相关的靶核酸上的靶序列杂交的间隔序列，和(ii)连接至所述间隔序列的能够引导所述工程化的Cas12i效应蛋白结合至所述crRNA以形成靶向所述靶序列的CRISPR-Cas12i复合物的直接重复序列；其中所述间隔序列和所述靶序列的杂交介导所述工程化的Cas12i效应蛋白与所述靶序列接触，导致所述工程化的Cas12i效应蛋白对所述靶序列进行修饰(例如切割或碱基编辑)，从而治疗所述受试 者的疾病。其中，所述CRISPR-Cas12i系统的任一组分可以同时递送，依次递送、或以DNA/RNA、DNA/DNA、RNA/RNA、蛋白/RNA、或蛋白/DNA任一形式递送。
进一步提供了使用根据本文所述的修饰细胞中的靶核酸的方法中的任一种的治疗方法。在一些实施方案中，本申请提供了治疗与个体细胞中的靶核酸相关的疾病或病况的方法，其包括使所述靶核酸与本文所述工程化的CRISPR-Cas12i系统中任一种接触，其中所述指导RNA的指导序列与所述靶核酸的靶序列互补，其中所述工程化的Cas12i效应蛋白和所述指导RNA彼此缔合以结合至所述靶核酸以修饰(如切割或碱基替换)所述靶核酸，从而使所述疾病或病况得到治疗。在一些实施方案中，将突变(例如，敲除或敲入突变)引入所述靶核酸。在一些实施方案中，所述靶核酸的表达被增强。在一些实施方案中，所述靶核酸的表达被抑制。在一些实施方案中，本申请提供了治疗个体的疾病或病况的方法，其包括向所述个体施用有效量的本文所述工程化的CRISPR-Cas12i系统中任一种以及编码治疗剂的供体DNA(如非致病的天然序列用作修复模板)，其中所述指导RNA的指导序列与所述个体的靶核酸的靶序列互补，其中所述工程化的Cas12i效应蛋白和所述指导RNA彼此结合以结合至所述靶核酸并将供体DNA插入所述靶序列中，从而使所述疾病或病况得到治疗。
在一些实施方案中，本申请提供了治疗个体的疾病或病况的方法，其包括向所述个体施用有效量的包含经修饰的靶核酸的工程化的细胞，其中所述工程化的细胞是通过使所述细胞与本文所述工程化的CRISPR-Cas12i系统中任一种接触而制备的，其中所述指导RNA的指导序列与所述靶核酸的靶序列互补，其中所述工程化的Cas12i效应蛋白和所述指导RNA相互缔合以结合至靶核酸以修饰所述靶核酸。在一些实施方案中，所述工程化的细胞是免疫细胞。在一些实施方案中，所述工程化的细胞是干细胞(如造血干细胞、神经干细胞)。在一些实施方案中，所述工程化的细胞是神经细胞。在一些实施方案中，所述个体是人。在一些实施方案中，所述个体是动物，例如模型动物诸如啮齿动物、宠物或农场动物。在一些实施方案中，所述个体是哺乳动物，如猫、狗、兔、仓鼠等。
在一些实施方案中，所述疾病或病况选自下组：癌症、心血管疾病、遗传性疾病、自身免疫性疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。在一些实施方案中，所述靶核酸是PCSK9。在一些实施方案中，所述疾病或病况是心血管疾病。在一些实施方案中，所述疾病或病况是冠状动脉疾病。在一些实施方案中，所述方法降低个体的胆固醇水平。在一些实施方案中，所述方法治疗个体的糖尿病。
本发明的CRISPR-Cas12i系统可以用于治疗的病症或疾病包括但不限于，囊性纤维化、遗传性血管水肿、糖尿病、进行性假肥大性肌营养不良、贝克肌营养不良、α-1-抗胰 蛋白酶缺乏、庞贝病、强直性肌营养不良、亨廷顿病、脆性X综合征、弗里德赖希共济失调、肌萎缩侧索硬化、额颞叶痴呆、遗传性慢性肾脏病、高脂血症、高胆固醇血症、莱伯氏先天性黑蒙、镰状细胞病或β地中海贫血等。在某些实施方式中，所述病症或疾病为转甲状腺素蛋白淀粉样变性，例如野生型转甲状腺素蛋白淀粉样变性(transthyretin-related wild-type amyloidosis，ATTRwt)、遗传性转甲状腺素蛋白淀粉样变性(transthyretin-related hereditary amyloidosis，ATTRm)、家族性淀粉样多发性神经病(familial amyloid polyneuropathy，FAP，ATTR-PN)、或家族性淀粉样变心肌病(familial amyloid cardiomyopathy，FAC，ATTR-CM)。在某些实施方式中，所述病症或疾病为TTR基因突变或异常表达(如高表达)引起的转甲状腺素蛋白不稳定。在某些实施方式中，所述病症或疾病为TTR基因突变或异常表达(如高表达)引起的其他病症或疾病，或衍生病症或疾病。
递送方法
在一些实施方案中，本文所述工程化的CRISPR-Cas12i系统或其组分、其核酸分子、或编码或提供其组分的核酸分子可通过多种递送系统诸如质粒或病毒递送至宿主细胞(例如，上面“构建体和载体”小节中描述的载体中的任一种)。在一些实施方案或方法中，所述工程化的CRISPR-Cas12i系统可以通过其他方法递送，如由所述工程化的Cas12i效应蛋白及其一个或多个同源RNA指导序列组成的核糖核蛋白复合物的核转染或电穿孔。
在一些实施方案中，通过纳米颗粒或外泌体来递送。
在一些实施方案中，成对的Cas12i切口酶复合物可以使用纳米颗粒或其他直接蛋白递送方法直接递送，使得包含两个成对的crRNA元件的复合物被共同递送。此外，蛋白可以通过病毒载体或直接地递送至细胞，然后直接递送包含针对双切口的两个成对间隔区的CRISPR阵列。在某些情况下，对于直接RNA递送，可以将RNA缀合于至少一个糖部分诸如N-乙酰基半乳糖胺(GalNAc)(特别是三触角GalNAc)。
在一些实施方案或方法中，所述工程化的CRISPR-Cas12i系统可以使用任何适合所治疗疾病的递送方法来递送，例如通过静脉注射或滴注方式进行递送，或患病部位局部递送(如肿瘤内递送)。施用本文所述工程化的CRISPR-Cas12i系统的合适途径包括但不限于：局部、皮下、经皮、皮内、病灶内、关节内、腹膜内、膀胱内、经粘膜、牙龈、牙内、耳蜗内、经鼓膜、器官内、硬膜外、鞘内、肌肉内、静脉内、血管内、骨内、眼周、瘤内、脑内和脑室内施用。在一些实施方案中，本文所述工程化的CRISPR-Cas12i系统通过注射、通过导管、通过栓剂或通过植入物向受试者施用，植入物为多孔、无孔或凝胶状材料，包括膜如唾液酸膜，或纤维。
IV.试剂盒和制品
还提供了包含本文所述工程化的Cas12i核酸酶或其功能变体、工程化的Cas12i效应蛋白或工程化的CRISPR-Cas12i系统中任一种的一种或多种组分的组合物、试剂盒、单位药剂和制品。
在一些实施方案中，提供了试剂盒，其包含：一个或多个AAV载体，其编码本文所述工程化的Cas12i核酸酶或其功能变体、工程化Cas12i效应蛋白或工程化CRISPR-Cas12i系统中任一种。在一些实施方案中，所述试剂盒还包含一种或多种指导RNA(或其编码DNA或载体)。在一些实施方案中，所述试剂盒还包含供体DNA。在一些实施方案中，所述试剂盒还包含细胞诸如人细胞。
所述试剂盒可包含一种或多种额外组分，诸如容器、试剂、培养基、细胞因子、缓冲液、抗体等，以允许工程化的细胞的繁殖。所述试剂盒还可包含用于施用组合物的装置。
所述试剂盒还可包含使用本文所述工程化的CRISPR-Cas12i系统的说明书，诸如检测或修饰靶核酸的方法。在一些实施方案中，所述试剂盒包含用于处理或诊断疾病或病况的说明书。与所述试剂盒组分使用有关的说明书通常包括针对该刻意处理的给药量、给药时间表和给药途径的信息。所述容器可以是单位剂量、散装包装(例如多剂量包装)或亚单位剂量。例如，可以提供这样的试剂盒(其包含足够剂量的本文所公开的组合物)以在延长的时期内提供对个体的有效处理。试剂盒还可以包括多个单位剂量的组合物和使用说明书，其包装数量足以在药房(例如医院药房和复方药房)中存储和使用。
本发明的试剂盒处于合适的包装中。合适的包装包括但不限于：小瓶、瓶子、广口瓶、软包装(例如密封的聚酯薄膜或塑料袋)等。试剂盒可以任选地提供额外的组分，如缓冲液和解释性信息。因此，本申请还提供了一种制品，其包括小瓶(如密封的小瓶)、瓶子、广口瓶、柔性包装等。
所述制品可以包括容器以及在容器上或与容器粘合的标签或包装插页。合适的容器包括例如瓶子、小瓶、注射器等。所述容器可以由多种材料(诸如玻璃或塑料)形成。通常，所述容器容纳有效处理本文所述的疾病或病症的组合物，并且可以具有无菌进入口(例如，容器可以是静脉注射溶液袋或具有可通过皮下注射针刺穿的塞子的小瓶)。所述标签或包装插页表明该组合物用于处理个体中的特定病况。所述标签或包装插页将进一步包括用于将组合物向个体施用的说明书。
包装插页是指通常包含在治疗产品的商业包装中的说明，所述说明包含关于使用此类治疗产品的适应症、用法、给药量、施用、禁忌症和/或警告的信息。
此外，所述制品还可包括第二容器，其包含药学上可接受的缓冲液，如抑菌注射用水(BWFI)、磷酸盐缓冲盐水、林格氏溶液和葡萄糖溶液。从商业和用户的角度来看，它还可以包括其他材料，包括其他缓冲液、稀释剂、过滤器、针头和注射器。必要时，还可包括增溶剂和局部麻醉剂(如，利多卡因)，以缓解注射部位的疼痛。
通常，成分以单位剂型单独提供或混合在一起提供，例如，作为显示活性剂量的密封容器如安瓿或小袋中的干燥冻干粉或无水浓缩物。当药物通过输液施用时，可用装有无菌药用级水或盐水的输液瓶进行配药。当药物组合物通过注射施用时，可提供无菌注射用水或盐水的安瓿，以便在施用前可混合成分。
实施例部分
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
实施例1：将参比Cas12i2酶中与PAM相互作用的氨基酸替换为带正电的氨基酸，并验证其基因编辑效率。
质粒构建
Cas12i2的编码序列经过密码子优化(人类)并合成。通过基于PCR的定点诱变产生Cas蛋白的变体。具体的方法是以突变的位点为中心将Cas12i2蛋白的DNA序列设计分成两部分，设计两对引物分别扩增这两部分DNA序列，同时引物上引入需要突变的序列，最后通过Gibson克隆的方式将两个片段装载到pCAG-2A-eGFP载体(带有青霉素抗性)上。突变体的组合则通过将Cas12i2蛋白的DNA拆分成多段，使用PCR、Gibson clone实现构建。突变体的位置确定使用本领域常用的蛋白质结构可视化软件(例如PyMol、Chimera等软件都可以选用)分析Cas12i2的结构信息而获得的。Cas12i2的结构信息参照PDB：6LTU，6LTR，6LU0，6LTP)。Cas12i2效应蛋白通过pCAG-2A-eGFP载体在人293T细胞中表达。将编码Cas12i2蛋白的DNA插入XmaI和NheI之间。通过将含有靶序列的退火寡核苷酸连接到BasI消化的pUC19-U6-i2-crRNA骨架中构建了在293T中表达Cas12i2的crRNA的载体。编码DR的核苷酸序列为SEQ ID NO：59。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24孔-细胞培养皿(Corning)中16小时，直到细胞密 度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas12i2蛋白的质粒和300ng编码crRNA的质粒转染到每个24孔-细胞培养皿中。转染约72h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化待荧光激活细胞分选(FACS)的HEK293T细胞。使用具有GFP通道的MoFlo XDP(Beckman Coulter)进行细胞分选。
靶向深度测序分析以进行基因组修饰
将FACS分选的GFP-阳性HEK293T细胞用缓冲液L裂解，并在55℃下孵育3小时，然后在95℃下孵育10分钟。使用相应的引物对在不同基因组位点中包含靶位点的dsDNA片段进行PCR扩增。对于靶向的深度测序，直接使用细胞裂解液作为模板，通过条形码(barcoded)PCR直接扩增靶位点。纯化PCR产物并归集到几个文库中以进行高通量测序。通过计算包含插入或缺失的读取(reads)的比率，采用CRISPResso2软件分析插入缺失的频率(％)。在本申请中，统一使用插入缺失的频率(％)这一指标来进行基因编辑效率的对比和分析。数量少于完整读取的0.05％的读取被废弃。
实施例1-A选出了四个具有单个氨基酸替换的工程化Cas12i2
按照实施例1描述的方法分别表达氨基酸序列具有单个突变的工程化Cas12i2酶，优选的氨基酸替换方式及其相应的基因编辑效率的见于图1a、图1b和表1。我们首先选取了Cas12i2距离PAM DNA
Figure PCTCN2022095072-appb-000001
以内的10个氨基酸：E176、E178、Y226、A227、N229、E237、K238、K264、T447、E563，进行精氨酸(R)的点突变测试。如图1a和表1所示，通过在293T细胞中比较这些Cas12i突变体与野生型Cas12i2(SEQ ID NO：1)在2个基因组位点：CCR5-3、RNF2-7的基因编辑效率，带有下述氨基酸替换的Cas12i突变体：E176R、K238R、T447R和E563R能够有效地提高基因编辑效率(图1a中显示，E176R、K238R、T447R和E563R四种单氨基酸替换都获得了高于约10％(此为针对CCR5-3的参比酶的基因编辑效率)以及高于约12％(此为针对RNF2-7的参比酶的基因编辑效率)插入缺失率，明显优于其他的单个氨基酸替换方案)，其余6种突变体则对基因编辑效率的提高没有帮助，甚至降低。在此所使用的针对CCR5-3和针对RNF2-7的crRNA间隔序列的编码序列分别如SEQ ID NO：60和61所示。
实施例1-B比较同时具有多个优选的氨基酸替换的工程化Cas12i2
按照实施例1描述的方法分别表达其氨基酸序列同时具有两个以上优选的氨基酸替换的工程化Cas12i2酶，其组合方式及其基因编辑效率的对比见于表1和图1b。我们将由实施例1-A筛选得到的E176R、K238R、T447R和E563R这4个能够提高效率的突变体中的点突变进行组合。如图1b和表1所示，通过在293T细胞中比较这些突变体与野生型Cas12i2在3个基因组位点：CCR5-3，CCR5-5，、RNF2-7的基因编辑效率，我们发现 点突变组合之后能够获得效率更进一步提升的突变体。尤其是当把4种突变组合到一起(E176R+K238R+T447R+E563R)时能够获得效率最优的组合突变。在此所使用的针对CCR5-3、针对RNF2-7以及针对CCR5-5的crRNA间隔序列的编码序列分别如SEQ ID NO：60、61和62所示。
表1实施例1的结果(基因编辑效率)总结
Figure PCTCN2022095072-appb-000002
实施例2：将参比Cas12i2酶中参与打开DNA双链的氨基酸替换为带芳香环的氨基酸，并分别验证它们的基因编辑的效率
质粒构建
通过基于PCR的定点诱变产生Cas12i2蛋白的变体，具体的方法是以突变的位点为中心将Cas12i2蛋白的DNA序列设计分成两部分，设计两对引物分别扩增这两部分DNA序列，同时引物上引入需要突变的序列，最后通过Gibson clone的方式将两个片段装载到pCAG-2A-eGFP载体上。氨基酸替换位置的确定可以使用常见的蛋白质结构可视化软件(例如，可以采用PyMol、Chimera等软件)分析Cas12i2的结构信息得到。Cas12i2的结构信息参照PDB：6LTU，6LTR，6LU0，6LTP)。Cas12i2效应蛋白通过pCAG-2A-eGFP载体在人293T细胞中表达。将编码Cas12i2蛋白的DNA插入XmaI和NheI之间。通过将含有靶序列的退火寡核苷酸连接到BasI消化的pUC19-U6-i2-crRNA骨架中构建了在293T中表达Cas12i2crRNA的载体。编码DR的核苷酸序列为SEQ ID NO：59。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24孔-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas12i2蛋白的质粒和300ng编码crRNA的质粒转染到每个24孔-细胞培养皿中。转染68后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化待荧光激活细胞分选(FACS)的HEK293T细胞。使用具有GFP通道的MoFlo XDP(Beckman Coulter)进行细胞分选。
靶向深度测序分析以进行基因组修饰
将FACS分选的GFP-阳性HEK293T细胞用缓冲液L裂解，并在55℃下孵育3小时，然后在95℃下孵育10分钟。使用相应的引物对在不同基因组位点中包含靶位点的dsDNA片段进行PCR扩增。对于靶向的深度测序，直接使用细胞裂解液作为模板，通过条形码(barcoded)PCR直接扩增靶位点。纯化PCR产物并归集到几个文库中以进行高通量测序。通过计算包含插入或缺失的读取(reads)的比率，采用CRISPResso2软件分析插入缺失的频率(％)。数量少于完整读取的0.05％的读取被废弃。
首先我们选取了Cas12i2酶中参与打开DNA双链的氨基酸：Q163和N164进行带有芳香环的氨基酸(Y，F，W)的点突变测试。从图2和表2可以看出，通过在293T细胞中比较这些突变体与野生型Cas12i2在3个基因组位点：CCR5-3，CCR5-5，RNF2-7的基因编辑效率，我们发现，有5个突变体：Q163W，Q163Y，Q163F，N164Y，N164F能够有效地提高基因编辑效率(至少在一个基因组位点)。特别地，N164Y以及N164F在3个基因组位点都展示了极佳的基因编辑效率。N164W相比于参比酶没有改善基因编辑效率的效果。
表2实施例2的结果(基因编辑效率)总结
Figure PCTCN2022095072-appb-000003
实施例3：将参比Cas12i2酶中位于RuvC结构域并与单链DNA底物相互作用的氨基酸替换为带正电的氨基酸，并验证其基因编辑的效率
质粒构建
通过基于PCR的定点诱变产生Cas12i2蛋白的变体，具体的方法是以突变的位点为中心将Cas12i2蛋白的DNA序列设计分成两部分，设计两对引物分别扩增这两部分DNA序列，同时引物上引入需要突变的序列，最后通过Gibson clone的方式将两个片段装载到pCAG-2A-eGFP载体上。突变体的组合则通过将Cas12i2蛋白的DNA拆分成多段，使用PCR、Gibson clone实现构建。突变体的位置确定是用常用的蛋白质结构可视化软件(例如，PyMol、Chimera等软件均可采用)分析Cas12i2的结构信息得到的。Cas12i2的结构信息参照PDB ID：6LTU，6LTR，6LU0，6LTP。这些Cas12i2结构中显示的ssDNA底物只有5nt。为了得到更长的ssDNA与Cas12i2相互作用的信息，我们将Cas12i1的结构(PDB ID：6W5C，6W62和6W64；Zhang H.et al.Nature Structural&Molecular Biology 27，1069-1076(2020))与Cas12i2的结构做了同源比对，以便将该Cas12i1结构中的ssDNA底物(9nt)放入了Cas12i2的RuvC催化口袋，通过该模型进一步寻找9A之内的氨基酸。Cas12i2效应蛋白通过pCAG-2A-eGFP载体在人293T细胞中表达。将编码Cas12i2蛋白的DNA插入XmaI和NheI之间。通过将含有靶序列的退火寡核苷酸连接到BasI消化的pUC19-U6-i2-crRNA骨架中构建了在293T中表达Cas12i2crRNA的载体。编码DR的核苷酸序列为SEQ ID NO：59。针对CCR5-3和针对RNF2-7的crRNA间隔序列的编码序列分别如SEQ ID NO：60和61所示。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24孔-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas12i2蛋白的质粒和300ng编码crRNA的质粒转染到每个24孔-细胞培养皿中。转染68h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化待荧光激活细胞分选(FACS)的HEK293T细胞。使用具有GFP通道的MoFlo XDP(Beckman Coulter)进行细胞分选。
靶向深度测序分析以进行基因组修饰
将FACS分选的GFP-阳性HEK293T细胞用缓冲液L裂解，并在55℃下孵育3小时，然后在95℃下孵育10分钟。使用相应的引物对在不同基因组位点中包含靶位点的dsDNA片段进行PCR扩增。对于靶向的深度测序，直接使用细胞裂解液作为模板，通过条形码(barcoded)PCR直接扩增靶位点。纯化PCR产物并归集到几个文库中以进行高通量测序。通过计算包含插入或缺失的读取(reads)的比率，采用CRISPResso2软件分析插入缺失的频率(％)。数量少于完整读取的0.05％的读取被废弃。
我们将参比Cas12i2酶中位于RuvC结构域并与单链DNA底物相互作用的氨基酸替换为 带正电的氨基酸。在293T细胞中比较这些突变体与野生型Cas12i2在CCR5-3和/或RNF2-7基因组位点的基因编辑效率。如图3a和表3所示，N391R、I926R、G929R能够有效地提高基因编辑效率(至少在一个基因组位点)。其中I926R在2个基因组位点都展示了极佳的基因编辑效率。如图3b、3c和表3所示，许多突变体能够有效地提高基因编辑效率(至少在一个基因组位点)。其中基因编辑效率提高的单个氨基酸替换方案的排名为：D362R＞E323R＞Q425R＞N925R＞其他效率提高的突变体。
我们将图3a、3b、3c筛选得到的E323R、D362R、Q425R和I926R这4个能够提高效率的突变体中的点突变进行组合。如图3d和表3所示，通过在293T细胞中比较这些突变体与野生型Cas12i2在2个基因组位点：CCR5-3，RNF2-7的基因编辑效率，我们发现点突变组合之后能够获得基因编辑效率更进一步提升的突变体(至少在一个位点)。
我们将图3a、3b、3c、3d筛选得到的部分能够提高效率的突变体中的点突变或组合与柔性区突变I926G、L439(L+GG)进行组合。如图3e和表3所示，通过在293T细胞中比较这些突变体与野生型Cas12i2在RNF2-7位点的基因编辑效率，我们发现点突变组合之后能够获得效率更进一步提升的突变体，例如N925R+I926G、I926R+L439(L+GG)。由此可见，进一步引入柔性区突变可以增加Cas12i突变体的基因编辑效率。
表3实施例3的结果(基因编辑效率)总结
Figure PCTCN2022095072-appb-000004
Figure PCTCN2022095072-appb-000005
*L439(L+GG)的含义是，在439号氨基酸后插入两个甘氨酸。
实施例4：将参比Cas12i2酶中与DNA-RNA双螺旋相互作用的氨基酸替换为带正电的氨基酸，并验证其基因编辑的效率
质粒构建
通过基于PCR的定点诱变产生Cas12i2蛋白的变体，具体的方法是以突变的位点为中心将Cas12i2蛋白的DNA序列设计分成两部分，设计两对引物分别扩增这两部分DNA序列，同时引物上引入需要突变的序列，最后通过Gibson clone的方式将两个片段装载到pCAG-2A-eGFP载体上。突变体的组合则通过将Cas12i2蛋白的DNA拆分成多段，使用PCR、Gibson clone实现构建。突变体的位置确定是用常用的蛋白质结构可视化软件(例如，PyMol、Chimera等软件均可采用)分析Cas12i2的结构信息得到的。Cas12i2的结构信息参照PDB：6LTU，6LTR，6LU0，6LTP)。Cas12i2效应蛋白通过pCAG-2A-eGFP载体在人293T细胞中表达。将编码Cas12i2蛋白的DNA插入XmaI和NheI之间。通过将含 有靶序列的退火寡核苷酸连接到BasI消化的pUC19-U6-i2-crRNA骨架中构建了在293T中表达Cas12i2crRNA的载体。编码DR的核苷酸序列为SEQ ID NO：59。针对CCR5-3和针对RNF2-7的crRNA间隔序列的编码序列分别如SEQ ID NO：60和61所示。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24孔-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas12i2蛋白的质粒和300ng编码crRNA的质粒转染到每个24孔-细胞培养皿中。转染68h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化待荧光激活细胞分选(FACS)的HEK293T细胞。使用具有GFP通道的MoFlo XDP(Beckman Coulter)进行细胞分选。
靶向深度测序分析以进行基因组修饰
将FACS分选的GFP-阳性HEK293T细胞用缓冲液L裂解，并在55℃下孵育3小时，然后在95℃下孵育10分钟。使用相应的引物对在不同基因组位点中包含靶位点的dsDNA片段进行PCR扩增。对于靶向的深度测序，直接使用细胞裂解液作为模板，通过条形码(barcoded)PCR直接扩增靶位点。纯化PCR产物并归集到几个文库中以进行高通量测序。通过计算包含插入或缺失的读取(reads)的比率，采用CRISPResso2软件分析插入缺失的频率(％)。数量少于完整读取的0.05％的读取被废弃。
图4和表4总结了在293T细胞中比较本实施例中的Cas12i2突变体与野生型Cas12i2在2个基因组位点：CCR5-3、RNF2-7的基因编辑效率。我们发现，有7个突变体：G116R、E117R、T159R、S161R、E319R、E343R、和D958R能够有效地提高基因编辑效率(至少在一个基因组位点)。其中D958R在2个基因组位点都展示了极佳的基因编辑效率。
表4实施例4的结果(基因编辑效率)总结
Figure PCTCN2022095072-appb-000006
Figure PCTCN2022095072-appb-000007
实施例5：将实施例1-4中筛选得到的部分提高基因编辑效率Cas12i2工程化改造氨基酸突变进行组合，并验证它们的基因编辑的效率。
质粒构建
突变体的组合则通过将Cas12i2蛋白的DNA拆分成多段，使用PCR、Gibson clone实现构建。突变体的位置确定是使用常用蛋白质结构可视化软件(例如，PyMol、Chimera等软件都可以采用)分析Cas12i2的结构信息得到的。Cas12i2的结构信息参照PDB：6LTU，6LTR，6LU0，6LTP)。Cas12i2效应蛋白通过pCAG-2A-eGFP载体在人293T细胞中表达。将编码Cas12i2蛋白的DNA插入XmaI和NheI之间。通过将含有靶序列的退火寡核苷酸连接到BasI消化的pUC19-U6-i2-crRNA骨架中构建了在293T中表达Cas12i2crRNA的载体。编码DR的核苷酸序列为SEQ ID NO：59。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24孔-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas12i2蛋白的质粒和300ng编码crRNA的质粒转染到每个24孔-细胞培养皿中。转染约68h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化待荧光激活细胞分选(FACS)的HEK293T细胞。使用具有GFP通道的MoFlo XDP(Beckman Coulter)进行细胞分选。
靶向深度测序分析以进行基因组修饰
将FACS分选的GFP-阳性HEK293T细胞用缓冲液L裂解，并在55℃下孵育3小时，然后在95℃下孵育10分钟。使用相应的引物对在不同基因组位点中包含靶位点的dsDNA片段进行PCR扩增。对于靶向的深度测序，直接使用细胞裂解液作为模板，通过条形码(barcoded)PCR直接扩增靶位点。纯化PCR产物并归集到几个文库中以进行高通量测序。通过计算包含插入或缺失的读取(reads)的比率，采用CRISPResso2软件分析插入缺失的频率(％)。数量少于完整读取的0.05％的读取被废弃。
我们将实施例1-4中筛选得到的氨基酸突变或者氨基酸突变组合：E176R+K238R+T447R+E563R，N164Y，E323R+D362R，I926R，E323R+D362R+I926R，E323R+D362R+I926G，E323R+D362R+I926G+L439(L+G)，E323R+D362R+I926G+L439(L+GG)进行进一步的组合。如图5和表5所示，通过在293T细胞中比较这些突变体与野生型Cas12i作用于5个基因组位点：CCR5-3，CCR5-5，CD34-8，CD34-9，RNF2-14的基因编辑效率，我们发现不同种类的点突变组合之后能够获得效率更进一步提升的突变体。同时，将我们认为效率最优的突变体(E176R+K238R+T447R+E563R+N164Y+E323R+D362R)命名为CasXX。在此，所使用的针对CD34-8、CD34-9以及RNF2-14的crRNA间隔序列的编码序列分别如SEQ ID NO：63、64和65所示。
表5实施例5的结果(基因编辑效率)总结(仅以RNF2-14处体现的基因编辑效率数据为例)
Figure PCTCN2022095072-appb-000008
Figure PCTCN2022095072-appb-000009
*L439(L+G)的含义是，在439号氨基酸后插入一个甘氨酸；在本说明书的上下文以及附图中，有时也表示为439G。
此外，我们还构建了以下突变组合：E176R+K238R+T447R+E563R+N164Y+D958R；
E176R+K238R+T447R+E563R+I926R+D958R；
E176R+K238R+T447R+E563R+E323R+D362R+D958R；
N164Y+I926R+D958R；N164Y+E323R+D362R+D958R；
E176R+K238R+T447R+E563R+N164Y+I926R+D958R；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+D958R；
E176R+K238R+T447R+E563R+N164Y+I926R+E323R+D362R+D958R；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+D958R；
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+L439(L+GG)+D958R；和
E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+L439(L+G)+D958R。可以通过T7核酸内切酶1(T7E1)测定和靶向深度测序检测其基因编辑效率。
实施例6：CasXX与常规基因编辑工具的比较验证其基因编辑效率。
质粒构建
为比较CasXX和其他Cas的基因编辑活性，AsCas12a，BhCas12b v4，SpCas9，SaCas9，SaCas9-KKH的编码序列经过密码子优化(人类)并合成。Cas效应蛋白通过pCAG-2A-eGFP载体在人293T细胞中表达。将编码Cas蛋白的DNA插入XmaI和NheI之间。通过将含有靶序列的退火寡核苷酸连接到BasI消化的pUC19-U6-i2-crRNA骨架中，构建了在293T中表达AsCas12a，BhCas12b v4，SpCas9，SaCas9，SaCas9-KKH和Cas12i2的sgRNA或crRNA的载体。编码CasXX的DR的核苷酸序列为SEQ ID NO：59。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的 DMEM(Gibco)中培养。将细胞接种在24孔-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas12i2蛋白的质粒和300ng编码crRNA的质粒转染到每个24孔-细胞培养皿中。转染约68h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化待荧光激活细胞分选(FACS)的HEK293T细胞。使用具有GFP通道的MoFlo XDP(Beckman Coulter)进行细胞分选。
靶向深度测序分析以进行基因组修饰
将FACS分选的GFP-阳性HEK293T细胞用缓冲液L裂解，并在55℃下孵育3小时，然后在95℃下孵育10分钟。使用相应的引物对在不同基因组位点中包含靶位点的dsDNA片段进行PCR扩增。对于靶向的深度测序，直接使用细胞裂解液作为模板，通过条形码(barcoded)PCR直接扩增靶位点。纯化PCR产物并归集到几个文库中以进行高通量测序。通过计算包含插入或缺失的读取(reads)的比率，采用CRISPResso2软件分析插入缺失的频率(％)。数量少于完整读取的0.05％的读取被废弃。
我们首先测试了CasXX在包含不同PAM序列的62个人基因组位点的基因编辑效率。设计的间隔序列为20个核苷酸。如图6a所示，CasXX展现了极其强大的基因编辑能力，平均基因编辑效率超过60％，并且几乎在测试的所有位点的基因编辑效率都超过了50％。并且，对于任意的NTTN PAM(N＝A，T，G，C)都展现了很高的基因编辑效率。
为了进一步展示工程化改造的CasXX的基因编辑能力，我们将CasXX与AsCas12a在TTTN PAM位点进行比较，以及与BhCas12b v4在TTN PAM位点进行比较。如图6b所示，CasXX在带有两种PAM的靶位点均展现了更高的平均基因编辑效率。
我们还将CasXX与SpCas9、SaCas9、SaCas9-KKH在相同位点进行比较。如图6c所示，CasXX在测试位点均展现了更高的平均基因编辑效率。
为测试CasXX在体内的基因编辑活性，编码CasXX的pCAG-2A-eGFP载体以及编码crRNA的pUC19-U6-i2-crRNA载体用脂质体转染法转染至小鼠Hepa1-6肝癌细胞系。其中，分别对65个内源基因位点设计了相应crRNA。设计的spacer序列为20个核苷酸。插入缺失频率由PCR扩增测序所得，和外源基因编辑分析方法类似。
如图6d所示，CasXX对小鼠Hepa1-6细胞系的65个内源基因位点展示出强大的基因编辑能力，平均基因编辑效率超过60％。
实施例7：使用CasXX在含有不同PAM的基因组位点进行基因编辑。
质粒构建
将编码CasXX的DNA序列装载到pCAG-2A-EGFP质粒中，从而构建了表达 CasXX质粒。通过将含有靶序列的退火寡核苷酸连接到BasI消化的pUC19-U6-crRNA骨架中构建了用于在HEK293T中表达crRNA的载体。其中，设计了64个不同的crRNA靶向64个人内源位点。所述内源靶核酸的5‘端有如图7所示不同PAM 5’-NNNN-3’(N＝A、T、G或C)，以检测CasXX对不同PAM的识别能力。其中，这64个位点包含的PAM序列覆盖了所有NNNN的组合：NTTN，NTAN，NTCN，NTGN，NATN，NAAN，NACN，NAGN，NCTN，NCAN，NCCN，NCGN，NGTN，NGAN，NGCN，NGGN。编码DR的核苷酸序列为SEQ ID NO：59。设计的间隔序列为20个核苷酸。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas蛋白的质粒和300ng编码crRNA的质粒转染到24孔细胞培养皿培养的细胞中。转染72h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化细胞，然后进行GFP荧光激活细胞分选(FACS)。
靶向深度测序分析以进行基因组修饰
将FACS分选的GFP-阳性293FT细胞用缓冲液L裂解，并在55℃下孵育3小时，然后在95℃下孵育10分钟。使用相应的引物对在不同基因组位点中包含靶位点的dsDNA片段进行PCR扩增。对于靶向的深度测序，直接使用细胞裂解液作为模板，通过条形码(barcoded)PCR直接扩增靶位点。纯化PCR产物并归集到几个文库中以进行高通量测序。通过计算包含插入或缺失的读取(reads)的比率，采用CRISPResso2软件分析插入缺失的频率(％)。数量少于完整读取的0.05％的读取被废弃。实验结果如图7所示。
如图7所示，CasXX在5’端带有NTTN，NTAN，NTCN，NTGN，NATN，NAAN，NCTN，NCAN，NGTN这些PAM的靶位点都展现了高效的基因编辑效率，在这些位点的平均基因编辑效率超过40％。
实施例8：使用CasXX在体外切割含有不同PAM的双链DNA
构建突变体的表达质粒
我们将编码野生型Cas12i2(SEQ ID NO：1)以及CasXX(SEQ ID NO：8)的DNA序列装载到BPK2014质粒(带有氯霉素抗性)中，从而构建了原核表达Cas12i2以及CasXX蛋白的质粒。
蛋白质纯化
将BPK2014原核表达质粒转化到大肠杆菌菌株BL21(λDE3)(TransGen Biotech)中， 并将转化后的菌液涂布在含有氯霉素的固体LB上。将三个克隆挑取到5ml液体LB中并培养过夜。然后将细菌转移到3L液体LB中继续培养，直至OD600达到0.6～0.8。然后用IPTG(0.5mM)在16℃下诱导20小时。超速离心，收获表达Cas12i的细菌，重悬于裂解缓冲液(50mM Tris-HCl、pH7.5、300mM NaCl)中并通过超声破碎。离心后，首先用Ni柱纯化上清液中的Cas12i蛋白。简而言之，在与上清液一起温育后，Ni柱依次用添加了0mM、20mM和50mM咪唑的裂解缓冲液洗涤。然后，Cas12i蛋白被添加500mM咪唑的裂解缓冲液洗脱。然后将收集的样品加载到离子交换柱(CM Sepharose Fast Flow，GE)中。野生型Cas12i2和CasXX蛋白用储存缓冲液(20mM Tris-HCl、300mM NaCl、1mM TCEP、10％甘油、pH7.5)洗脱。蛋白质通过过滤器灭菌并储存在-80℃。
crRNA体外转录
编码DR的核苷酸序列为SEQ ID NO：59。合成含有T7启动子序列的寡核苷酸(命名为T7-F)和含有crRNA和T7启动子互补序列的寡核苷酸(命名为T7-12i-crRNA-R)，并在1xNEBufferTM2(NEB)中退火。这些寡核苷酸的序列列于表6。将退火产物用作模板，通过HiScribeTM T7 Quick High Yield RNA Synthesis Kit(NEB)生产crRNA。使用
Figure PCTCN2022095072-appb-000010
RNA Cleanup Kit(NEB)纯化转录的crRNA。
表6 crRNA引物序列
Figure PCTCN2022095072-appb-000011
Cas12i蛋白体外酶切
为了制备用于体外切割的线性dsDNA底物，首先将含有相同原型间隔区和不同PAM的靶标克隆到由EcoR1和HindIII处理的pUC19(带有青霉素抗性)中。5’带有PAM的靶标序列列于表7。然后将携带靶标的pUC19质粒通过SacI线性化，并使用DNA Clean&Concentrator(Zymo Research)进行纯化。对于体外切割实验，首先将400nM Cas12i蛋白与2μM crRNA在37℃下孵育15分钟。接下来，在含有1x NEBufferTM3.1(NEB)的10μl反应体系中，将Cas12i-crRNA RNP与150ng线性化靶DNA在37℃下反应40分钟。然后，用50mM EDTA终止反应，用RNase cocktail(Invitrogen)在37℃下消化RNA 15分钟。最后，用蛋白酶(NEB)在37℃下处理样品15分钟。通过在1.2％琼脂糖凝胶上电泳分离反应产物。
如图8所示，野生型Cas12i2蛋白仅仅对含有5‘-NTTN-3‘PAM的双链DNA具有部分切割的效率，但是对其余的PAM几乎没有切割活性。但是，CasXX蛋白对含有NTTN，NTAN，NTCN，NTGN，NATN，NAAN，NACN，NCTN，NCAN，NGTN，NGAN PAM的双链DNA都展现了高效的切割效率。CasXX几乎能完全切割含有NTTN，NTAN，NTCN， NATN，NAAN，NACN，NCTN，NCAN，NGTN PAM的双链DNA。因此，本发明所提供的工程化的Cas12i核酸酶能用于更广泛的基因编辑或治疗。
表7：5’PAM-靶标序列
SEQ ID NO：43 NTTN-靶标 CTTCTTCAACATATCCAAACAAAT
SEQ ID NO：44 NTAN-靶标 CTACTTCAACATATCCAAACAAAT
SEQ ID NO：45 NTCN-靶标 CTCCTTCAACATATCCAAACAAAT
SEQ ID NO：46 NTGN-靶标 CTGCTTCAACATATCCAAACAAAT
SEQ ID NO：47 NATN-靶标 CATCTTCAACATATCCAAACAAAT
SEQ ID NO：48 NAAN-靶标 CAACTTCAACATATCCAAACAAAT
SEQ ID NO：49 NACN-靶标 CACCTTCAACATATCCAAACAAAT
SEQ ID NO：50 NAGN-靶标 CAGCTTCAACATATCCAAACAAAT
SEQ ID NO：51 NCTN-靶标 CCTCTTCAACATATCCAAACAAAT
SEQ ID NO：52 NCAN-靶标 CCACTTCAACATATCCAAACAAAT
SEQ ID NO：53 NCCN-靶标 CCCCTTCAACATATCCAAACAAAT
SEQ ID NO：54 NCGN-靶标 CCGCTTCAACATATCCAAACAAAT
SEQ ID NO：55 NGTN-靶标 CGTCTTCAACATATCCAAACAAAT
SEQ ID NO：56 NGAN-靶标 CGACTTCAACATATCCAAACAAAT
SEQ ID NO：57 NGCN-靶标 CGCCTTCAACATATCCAAACAAAT
SEQ ID NO：58 NGGN-靶标 CGGCTTCAACATATCCAAACAAAT
实施例9：使用GUIDE-Seq检测CasXX的脱靶效应
质粒构建
我们将编码CasXX的DNA序列装载到pCAG-2A-EGFP质粒中，从而构建了表达CasXX质粒。通过将含有靶序列的退火寡核苷酸连接到BasI消化的pUC19-U6-crRNA骨架中构建了在293T(人肾上皮细胞系)中表达Cas蛋白crRNA的载体。其中crRNA包含能靶向EMX1-7内源位点的间隔序列(spacer；SEQ ID NO：77)。编码DR的核苷酸序列为SEQ ID NO：59。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas蛋白的质粒、300ng编码crRNA的质粒和10pmol的退火的双链DNA标签(序列见表8)，转染到24孔细胞培养皿培养的细胞中。转染72h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化细胞，然后进行GFP荧光激活细胞分选(FACS)。分选出的是成功表达Cas酶的细胞。
表8双链DNA标签的引物序列(P代表磷酸修饰，*代表硫代磷酸二酯键修饰)
SEQ ID NO：25 GUIDE-F1 5′-P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3′
SEQ ID NO：26 GUIDE-R1 5′-P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C-3′
全基因组范围检测CasXX的脱靶效应
使用
Figure PCTCN2022095072-appb-000012
MicroElute Genomic DNA Kit(Omega)试剂盒提取FACS分选的GFP-阳性293T细胞用的基因组。纯化好的基因组使用用Qubit定量。使用Covaris S220仪器，按照仪器推荐的程序将基因组打断到500bp左右。然后使用VAHTS Universal Pro DNA Library Prep Kit for Illumina(Vazyme)试剂盒进行DNA建库。建库流程参照参考文献(Tsai，SQ et al.“GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases，”Nat Biotechnol.2015；33(2)：187-197；其内容通过引用整体并入本文)。将建库产物进行高通量测序。分析测序结果，寻找潜在的脱靶位点。我们对CasXX在EMX1-7位点的脱靶效应进行了系统的分析，具体的分析结果见附图10。第一行序列为参考靶序列，下面的序列分别代表靶序列和脱靶序列以及双链DNA标签在靶序列和脱靶序列富集的reads数目。
从较高的脱靶数目可以看出，CasXX酶在哺乳动物细胞中的编辑特异性并非很理想，还需要进一步优化。
实施例10：在CasXX序列(SEQ ID NO：8)基础上引入新的氨基酸突变，检验该突变体特异性提高的特征(选择了两个靶向位点进行突变体筛选实验，即EMX1-7位点和RNF2-1位点)
筛选实验准备：构建突变体的表达质粒
在CasXX(包含基于SEQ ID NO：1的N164Y+E176R+K238R+E323R+D362R+T447R+E563R突变)的基础上，我们拟进一步引入新的氨基酸点突变来提高CasXX基因编辑的特异性，降低脱靶效应。我们基于CasXX的序列(SEQ ID NO：8)，使用带有突变碱基的引物对CasXX的DNA序列进行PCR，使用
Figure PCTCN2022095072-appb-000013
HiFi DNA Assembly Master Mix(NEB)试剂盒将纯化的PCR产物装载到pCAG-2A-EGFP质粒中，从而构建了表达引入相关的点突变的CasXX质粒。我们一共获得了26种基于CasXX序列、包含单个氨基酸突变的突变体，分别命名为CasXX-HF-1到HF-26。具体的突变方式见表9。其中，CasXX-HF-26将CasXX中的N164Y点突变恢复成野生型Cas12i2的原始氨基酸N，即Y164N(即删减了N164Y这个突变)。
表9：CasXX-HF突变体
Figure PCTCN2022095072-appb-000014
Figure PCTCN2022095072-appb-000015
构建靶向EMX1-7位点的crRNA的表达质粒
通过将含有靶序列的退火寡核苷酸使用T4ligase(NEB)连接到BasI(NEB)消化处理的pUC19-U6-crRNA骨架中，构建了在293T中表达Cas蛋白crRNA的载体。所述crRNA设计为靶向EMX1-7位点。具体序列见表10。编码DR的核苷酸序列为SEQ ID NO：59。
表10：编码靶向EMX1-7的spacer的DNA序列
序列号 crRNA靶向位点名称 spacer编码序列
SEQ ID NO：27 EMX1-7 TGGTTGCCCACCCTAGTCAT
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas蛋白的质粒和300ng编码crRNA的质粒转染到24孔细胞培养皿培养的细胞中。转染72h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化细胞，然后进行GFP荧光激活细胞分选(FACS)。
使用T7E1酶切方法检测Cas蛋白在EMX1-7靶位点以及脱靶位点的编辑效率
FACS分选的GFP阳性HEK293T细胞用40μL缓冲液L(bimake)裂解，在55℃下孵育3小时，然后在95℃下孵育10分钟。使用针对EMX1-7靶位点、EMX1-7脱靶位点1、EMX1-7脱靶位点2、和EMX1-7脱靶位点3(如图10)的引物对在不同基因组位点中含有靶位点或者脱靶位点的dsDNA片段进行PCR扩增，靶位点或者脱靶位点的序列见表11。之后，使用10μL PCR产物，进行再退火程序以形成异源双链dsDNA。然后，将混合物用1/10体积的NEBuffer TM 2.1和0.2μL T7 endonuclease I(NEB)在37℃下处理50分钟。通过～2.5％琼脂糖凝胶电泳分析消化产物。根据条带的灰度值计算插入缺失的比例(Indel，％)。每个Cas12i突变体在靶位点或者脱靶位点的插入缺失的比例值见表12，对应图11。实验结果表明，R857A，N861A，K807A，N848A，R715A，R719A，K394A，H357A，K844A这些基于CasXX序列(SEQ ID NO：8)的单点突变体(对应SEQ ID NO：14-22的工程化Cas12i核酸酶，见表9)能够有效地降低在脱靶位点EMX1-7-OT-1，EMX1-7-OT-2，EMX1-7-OT-3的插入缺失比例，具有更高的特异性。在实验结果中，我们把脱靶位点处插入缺失率的降低和靶位点处编辑效率基本不变(或升高)定义为编辑特异性的提高。
表11 EXM1-7靶位点和脱靶位点序列
Figure PCTCN2022095072-appb-000016
表12 CasXX-HF1至CasXX-HF-26在EMX1-7靶序列和脱靶序列编辑效率比较
Figure PCTCN2022095072-appb-000017
Figure PCTCN2022095072-appb-000018
构建靶向RNF2-1位点的crRNA的表达质粒
通过将含有靶序列的退火寡核苷酸使用T4ligase(NEB)连接到BasI(NEB)消化处理的pUC19-U6-crRNA骨架中，构建了在HEK293T中表达Cas蛋白crRNA的载体。最终的crRNA都靶向同-个RNF2-1位点，但是spacer序列有所不同。编码这些spacer的具体序列见表13。其中RNF2-1-FM代表crRNA中的spacer序列与RNF2-1位点完全匹配。RNF2-1-Mis-1/2代表crRNA中的spacer序列与RNF2-1位点在第1个和第2个碱基位置不匹配，在其余位置匹配。RNF2-1-Mis-5/6代表crRNA中的spacer序列与RNF2-1位点在第5个和第6个碱基位置不匹配，在其余位置匹配。RNF2-1-Mis-17/1代表crRNA中的spacer序列与RNF2-1位点在第17个和第18个碱基位置不匹配，在其余位置匹配。RNF2-1-Mis-19/20代表crRNA中的spacer序列与RNF2-1位点在第19个和第20个碱基位置不匹配，在其余位置匹配。在此，设置碱基不匹配的目的是为了模拟脱靶效应。编码DR的核苷酸序列为SEQ ID NO：59。
表13靶向RNF2-1的间隔序列的DNA编码序列
SEQ ID NO： crRNA名称 spacer编码序列
32 crRNA-RNF2-1-FM TACAGGAGGCAATAACAGAT
33 crRNA-RNF2-1-Mis-1/2 GCCAGGAGGCAATAACAGAT
34 crRNA-RNF2-1-Mis-5/6 TACATTAGGCAATAACAGAT
35 crRNA-RNF2-1-Mis-17/18 TACAGGAGGCAATAACCTAT
36 crRNA-RNF2-1-Mis-19/20 TACAGGAGGCAATAACAGCG
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas蛋白的质粒和300ng编码crRNA的质粒，转染到24孔细胞培养皿培养的细胞中。转染72h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化细胞，然后进行GFP荧光激活细胞分选(FACS)。分选出的是成功表达Cas酶的细胞。
使用T7E1酶切法检测Cas12i突变体在RNF2-1以及脱靶位点的编辑效率
FACS分选的GFP阳性HEK293T细胞用40μL缓冲液L(bimake)裂解，在55℃下孵育3小时，然后在95℃下孵育10分钟。使用RNF2-1引物对对RNF2-1位点dsDNA片段进行PCR扩增。之后，使用10μL PCR产物，进行再退火程序以形成异源双链dsDNA。然后，将混合物用1/10体积的NEBuffer TM 2.1和0.2μL T7 endonuclease I(NEB)在37℃下处理50分钟。通过～2.5％琼脂糖凝胶电泳分析消化产物。根据条带的灰度值计算插入缺失的比例(Indel，％)。每个Cas12i突变体在使用不同crRNA时在靶位点的插入缺失的比例值见表14，对应图12。实验结果表明，R857A，N861A，K807A，N848A，R715A，R719A，K394A，H357A，K844A这些基于CasXX序列的单点突变体(对应SEQ ID NOs：14-22的工程化Cas12i核酸酶，见表9)即便在使用模拟脱靶的crRNA-RNF2-1-Mis-1/2，crRNA-RNF2-1-Mis-5/6，crRNA-RNF2-1-Mis-17/18和crRNA-RNF2-1-Mis-19/20时依然能够有效降低插入缺失的比例。在实验结果中，我们把脱靶位点处插入缺失率的降低和靶位点处编辑效率基本不变(或提高)定义为编辑特异性的提高。
表14：不同CasXX-HF突变体在使用靶向靶序列和模拟脱靶spacer序列的crRNA对RNF2-1位点的编辑效率
Figure PCTCN2022095072-appb-000019
Figure PCTCN2022095072-appb-000020
实施例11：在CasXX序列基础上引入新的氨基酸突变的突变体
针对在CasXX序列基础上引入新的氨基酸突变的突变体，在荧光报告系统中筛选特异性提高的那些突变体，本实施例和实施例10的区别在于体现编辑效率的分析手段由琼脂糖凝胶电泳改为荧光。
构建靶向mCherry基因的crRNA的表达质粒
通过将含有靶序列的退火寡核苷酸使用T4ligase(NEB)连接到BasI(NEB)消化处理的pUC19-U6-crRNA骨架中，构建了在HEK293T中表达Cas蛋白crRNA的载体。最终的crRNA都靶向同一个mCherry位点，但是spacer序列有所不同。编码spacer的具体序列见表15。其中mCherry-FM代表crRNA中的spacer序列与mCherry位点完全匹配。crRNA-mCherry-Mis-1/2代表crRNA中的spacer序列与crRNA-mCherry位点在第1个和第2个碱基位置不匹配，在其余位置匹配。crRNA-mCherry-Mis-5/6代表crRNA中的spacer序列与crRNA-mCherry位点在第5个和第6个碱基位置不匹配，在其余位置匹配。crRNA-mCherry-Mis-19/20代表crRNA中的spacer序列与crRNA-mCherry位点在第19个和第20个碱基位置不匹配，在其余位置匹配。编码DR的核苷酸序列为SEQ ID NO：59。
表15：靶向mCherry及模拟脱靶的spacer编码序列
SEQ ID NO： crRNA靶向位点名称 Spacer编码序列
37 crRNA-mCherry-FM ACCTTGTAGATGAACTCGCC
38 crRNA-mCherry-Mis-1/2 CACTTGTAGATGAACTCGCC
39 crRNA-mCherry-Mis-5/6 ACCTGTTAGATGAACTCGCC
40 crRNA-mCherry-Mis-19/20 ACCTTGTAGATGAACTCGAA
细胞培养、转染
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas蛋白的质粒，300ng编码crRNA的质粒和100ng编码mCherry的质粒，转染到24孔细胞培养皿培养的细胞中。
使用流式分析的方法检测Cas蛋白在mCherry位点的编辑效率
转染72h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化细胞，然后用流式细胞仪分析，计算GFP，mCherry荧光的比例。mCherry的比例越低，意味着Cas蛋白编辑mCherry基因的效率越高。流程示意图以及mCherry编辑效率公式见附图13。
实验结果表明，R857A，R719A，K394A，K844A(分别对应SEQ ID NO：14、19、20、22)这些基于CasXX序列的单点突变体在使用模拟脱靶的crRNA-mCherry-Mis-1/2， crRNA-mCherry-Mis-5/6和crRNA-mCherry-Mis-19/20时编辑mCherry基因的效率明显降低，见附图14(在脱靶位点mCherry％越高，脱靶效率越低)。在此，我们把脱靶位点处插入缺失率的降低和靶位点处编辑效率基本不变(或升高)定义为编辑特异性的提高。
实施例12：在CasXX序列基础上引入氨基酸组合突变，在内源基因(组合)位点筛选特异性提高的突变体
筛选实验准备：构建突变体的表达质粒
基于实施例10和11，我们选取R857A，R719A，K394A，K844A这几个点突变进行进一步组合探究。我们基于CasXX的序列，使用带有突变碱基的引物对CasXX的DNA序列进行PCR，我们使用
Figure PCTCN2022095072-appb-000021
HiFi DNA Assembly Master Mix(NEB)试剂盒将纯化的PCR产物装载到pCAG-2A-EGFP质粒中，从而构建了带有组合突变的CasXX质粒。我们一共获得了11种基于CasXX序列(SEQ ID NO：8)、包含组合突变的突变体命名及
具体的突变方式见表16。
表16：CasXX-HF突变体
Figure PCTCN2022095072-appb-000022
构建靶向EMX1-7位点以及RNF2-1的crRNA的表达质粒
通过将含有靶序列的退火寡核苷酸使用T4ligase(NEB)连接到BasI(NEB)消化处理的pUC19-U6-crRNA骨架中，构建了在293T中表达Cas蛋白crRNA的载体。crRNA如 实施例10所述进行设计，靶向EMX1-7或RNF2-1，或模拟RNF2-1脱靶。Spacer编码序列见表10和13。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码Cas蛋白的质粒和300ng编码crRNA的质粒，转染到24孔细胞培养皿培养的细胞中。转染72h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化细胞，然后进行荧光激活细胞分选(FACS)。分选出的是成功表达Cas酶的细胞。
使用T7E1酶切方法检测Cas蛋白在靶位点以及脱靶位点的编辑效率
FACS分选的GFP阳性HEK293T细胞用40μL缓冲液L(bimake)裂解，在55℃下孵育3小时，然后在95℃下孵育10分钟。使用实施例10所述相应的引物对在不同基因组位点中含有靶位点或者脱靶位点的dsDNA片段进行PCR扩增，靶位点或者脱靶位点的序列见表11和13。之后，使用10μL PCR产物，进行再退火程序以形成异源双链dsDNA。然后，将混合物用1/10体积的NEBuffer TM 2.1和0.2μL T7 endonuclease I(NEB)在37℃下处理50分钟。通过～2.5％琼脂糖凝胶电泳分析消化产物。根据条带的灰度值计算插入缺失的比例(Indel，％)。实验结果表明，R857A，R719A，K394A，K844A，R719A/K844A(SEQ ID NO：23)以及R857A/K844A(SEQ ID NO：24)这些基于CasXX序列的突变体能够有效地降低在EMX1-7-OT-1，EMX1-7-OT-2，EMX1-7-OT-3脱靶位点的插入缺失比例，并且不牺牲在靶位点的编辑效率，见表17，对应图15。同时，R857A，R719A，K394A，K844A，R719A/K844A以及R857A/K844A这些基于CasXX序列的突变体在使用模拟脱靶的crRNA-RNF2-1-Mis-1/2，crRNA-RNF2-1-Mis-5/6，crRNA-RNF2-1-Mis-17/18和crRNA-RNF2-1-Mis-19/20时能够有效降低插入缺失的比例，并且不牺牲在靶位点的效率，见表18，对应图16。在此，我们把脱靶位点处插入缺失率的降低和靶位点处编辑效率基本不变(或升高)定义为编辑特异性的提高。
表17：不同CasXX-HF突变体对EMX1-7的靶向和脱靶编辑效率
Figure PCTCN2022095072-appb-000023
Figure PCTCN2022095072-appb-000024
表18：不同CasXX-HF突变体对RNF2-1的靶向和脱靶编辑效率
Figure PCTCN2022095072-appb-000025
本说明书中的SEQ ID NO：23和SEQ ID NO：24所示突变体(对应于表16中标出序列号的突变体)具有明显更高的特异性。
实施例13：使用GUIDE-Seq检测CasXX以及CasXX+K394A突变体的脱靶效应。
质粒构建
将编码CasXX(SEQ ID NO：8)以及CasXX+K394A突变体(SEQ ID NO：20)的DNA序列装载到pCAG-2A-EGFP质粒中，从而构建了表达CasXX或(CasXX+K394A)的质粒。通过将含有靶序列的退火寡核苷酸连接到BasI消化的pUC19-U6-crRNA骨架中构建了在HEK293T中表达Cas12i蛋白crRNA的载体。其中crRNA设计为包含能靶向CD34-7内源位点的间隔序列。编码DR的核苷酸序列为SEQ ID NO：59。编码spacer的核苷酸序列包含SEQ ID NO：78。
细胞培养、转染以及荧光激活细胞分选法(FACS)
将HEK293T细胞在含1％青霉素-链霉素(Gibco)和10％胎牛血清(Gibco)的DMEM(Gibco)中培养。将细胞接种在24-细胞培养皿(Corning)中16小时，直到细胞密度达到70％。通过使用Lipofectamine 3000(Invitrogen)，将600ng编码CasXX或(CasXX+K394A)蛋白的质粒、300ng编码crRNA的质粒和10pmol的退火的双链DNA标签(序列见表8)，转染到24孔细胞培养皿培养的细胞中。转染72h后，用胰蛋白酶-EDTA(0.05％)(Gibco)消化细胞，然后进行GFP荧光激活细胞分选。
全基因组范围检测Cas12i2突变体的脱靶效应
使用
Figure PCTCN2022095072-appb-000026
MicroElute Genomic DNA Kit(Omega)试剂盒提取FACS分选的GFP-阳性HEK293T细胞用的基因组。纯化好的基因组使用用Qubit定量。使用Covaris S220仪器，按照仪器推荐的程序将基因组打断到500bp左右。然后使用VAHTS Universal Pro DNA Library Prep Kit for Illumina(Vazyme)试剂盒进行DNA建库。建库流程参照参考文献(Tsai，SQ et al.“GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases，”Nat Biotechnol.2015；33(2)：187-197)。将建库产物进行高通量测序。分析测序结果，寻找潜在的脱靶位点。我们对CasXX和(CasXX+K394A)在CD34-7内源位点的脱靶效应进行了系统的分析，具体的分析结果见图17。第一行序列为参考靶序列，下面的序列分别代表靶序列和脱靶序列以及双链DNA标签在靶序列和脱靶序列富集的reads数目。图17说明CasXX在CD34-7位点存在脱靶效应，但是CasXX+K394A突变体在该位点没有脱靶效应。因此，在CasXX序列的基础上额外引入K394A点突变能够大幅度提高CasXX的保真度，使得CasXX具有高的特异性。
尽管以上结合附图对本发明的实施方案进行了描述，但本发明并不局限于上述的具体实施方案和应用领域，上述的具体实施方案仅仅是示意性的、指导性的，而不是限制性的。本领域的普通技术人员在本说明书的启示下和在不脱离本发明权利要求所保护的范围的情况下，还可以做出很多种的形式，这些均属于本发明保护之列。
序列表
Figure PCTCN2022095072-appb-000027
Figure PCTCN2022095072-appb-000028
Figure PCTCN2022095072-appb-000029
Figure PCTCN2022095072-appb-000030
Figure PCTCN2022095072-appb-000031

Claims (38)

  1. 一种工程化的Cas12i核酸酶；其包含一种或多种基于参比Cas12i核酸酶的突变，所述突变选自：
    (1)将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸；
    (2)将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸
    (3)将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸
    (4)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为带正电的氨基酸；和
    (5)将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸；
    优选地，所述参比Cas12i核酸酶为氨基酸序列为SEQ ID NO：1的野生型Cas12i2核酸酶。
  2. 如权利要求1所述的工程化的Cas12i核酸酶，其中，所述与PAM相互作用的一个或多个氨基酸是与PAM在三维结构上距离在9埃以内的氨基酸，优选，所述与PAM相互作用的一个或多个氨基酸是下述位置的一个或多个氨基酸：176、178、226、227、229、237、238、264、447和563，
    进一步优选，所述与PAM相互作用的一个或多个氨基酸是下述一个或多个氨基酸：E176、E178、Y226、A227、N229、E237、K238、K264、T447和E563，
    进一步优选，所述与PAM相互作用的一个或多个氨基酸是下述一个或多个氨基酸：E176、K238、T447和E563，
    其中，所述氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
  3. 如权利要求1或2所述的工程化的Cas12i核酸酶，其中，所述带正电的氨基酸是R，K或H。
  4. 如权利要求1-3中任一项所述的工程化的Cas12i核酸酶，其中，所述将参比Cas12i核酸酶中与PAM相互作用的一个或多个氨基酸替换为带正电的氨基酸是指如下替换中的一种或多种：E176R、K238R、T447R和E563R；
    优选，所述工程化的Cas12i核酸酶包含下述任何一种突变或突变组合：(1)E563R；(2)E176R、T447R、E176R和E563R；(3)K238R和E563R；(4)E176R、K238R和T447R；(5)E176R、K238R和E563R；(6)E176R、T447R和E563R；和(7)E176R、K238R、T447R和E563R；
    其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  5. 如权利要求1～4中任一项所述的工程化的Cas12i核酸酶，其中，所述参与打开DNA双链的一个或多个氨基酸是与PAM中相对于靶标链的3’端最后一个碱基对相互作用的氨基酸；
    优选，所述参与打开DNA双链的一个或多个氨基酸是下述位置的一个或多个氨基酸：163和164；
    进一步优选，所述参与打开DNA双链的一个或多个氨基酸是下述一个或多个氨基酸：Q163和N164；进一步优选，所述参与打开DNA双链的氨基酸为N164；
    其中，所述氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
  6. 如权利要求1-5中任一项所述的工程化的Cas12i核酸酶，其中，所述参与打开DNA双链的一个或多个氨基酸替换为带芳香环的氨基酸，所述带芳香环的氨基酸是F、Y或W，优选，所述带芳香环的氨基酸为F或Y。
  7. 如权利要求1-6中任一项所述的工程化的Cas12i核酸酶，其中，所述将参比Cas12i核酸酶中参与打开DNA双链的一个或多个氨基酸的替换为带芳香环的氨基酸包括以下一个或多个替换：Q163F、Q163Y、Q163W、和N164F；优选，所述Cas12i核酸酶包含N164Y或N164F突变；进一步优选，所述工程化的Cas12i核酸酶包含N164Y。
  8. 如权利要求1～7中任一项所述的工程化的Cas12i核酸酶，其中，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是在三维结构上与单链DNA底物距离在9埃以内的氨基酸；
    优选，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是下述位置的一个或多个氨基酸：323、327、355、359、360、361、362、388、390、391、392、393、414、417、418、421、424、425、650、652、653、696、705、708、709、751、752、755、840、848、851、856、885、897、925、926、928、929、932、1022；
    进一步优选，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是下述一个或多个氨基酸：E323、L327、V355、G359、G360、K361、D362、L388、N390、N391、F392、K393、Q414、L417、L418、K421、Q424、Q425、S650、E652、 G653、I696、K705、K708、E709、L751、S752、E755、N840、N848、S851、A856、Q885、M897、N925、I926、T928、G929、Y932、A1022；
    进一步优选，所述位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸是是下述一个或多个氨基酸：E323、D362、Q425、N925、I926、N391、Q424和G929；
    其中，所述氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
  9. 如权利要求1-8中任一项所述的工程化的Cas12i核酸酶，其中，将参比Cas12i核酸酶中位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸包括替换为R或K，优选，所述带正电的该氨基酸为R。
  10. 如权利要求1-9中任一项所述的工程化的Cas12i核酸酶，其中，将参比Cas12i核酸酶位于RuvC结构域并与单链DNA底物相互作用的一个或多个氨基酸替换为带正电的氨基酸是指包括如下替换中的一种或多种：E323R、D362R、N391R、Q424R、Q425R、N925R、I926R和G929R；
    优选，所述工程化的Cas12i核酸酶包含下述任何一种突变或突变组合：(1)E323R；(2)D362R；(3)Q425R；(4)N925R；(5)I926R；(6)E323R和D362R；(7)E323R和Q425R；(8)E323R和I926R；(9)Q425R和I926R；(10)D362R和I926R；(11)N925R和I926R；(12)E323R、D362R和Q425R；(13)E323R、D362R和I926R；(14)E323R、Q425R和I926R；(15)D362R、N925R和I926R；和(16)E323R、D362R、Q425R和I926R；
    其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  11. 如权利要求1～10中任一项所述的工程化的Cas12i核酸酶，其中，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是与DNA-RNA双螺旋在三维结构上距离在9埃以内的氨基酸；
    优选，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是下述位置的一个或多个氨基酸：116、117、156、159、160、161、247、293、294、297、301、305、306、308、312、313、316、319、320、343、348、349、427、433、438、441、442、679、683、691、782、783、797、800、852、853、855、861、865、957、958；
    进一步优选，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是下述一个或多个氨基酸：G116、E117、A156、T159、E160、S161、E247、G293、E294、N297、T301、I305、K306、T308、N312、F313、Q316、E319、Q320、E343、E348、E349、D427、K433、V438、N441、Q442、N679、E683、E691、D782、E783、E797、E800、M852、D853、L855、N861、Q865、S957、D958；
    进一步优选，所述与DNA-RNA双螺旋相互作用的一个或多个氨基酸是下述一个或多个氨基酸：G116、E117、T159、S161、E319、E343和D958；其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  12. 如权利要求1-11中任一项所述的工程化的Cas12i核酸酶，其中，将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为带正电的氨基酸包括替换为R或K，优选，所述带正电的氨基酸为R。
  13. 如权利要求1-12中任一项所述的工程化的Cas12i核酸酶，其中，将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个氨基酸替换为带正电的氨基酸包括替换为以下的一种或多种：G116R、E117R、T159R、S161R、E319R、E343R和D958R；
    优选，所述工程化的Cas12i核酸酶包含D958R替换；
    其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  14. 如权利要求1～13中任一项所述的工程化的Cas12i核酸酶，所述与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸选自如下的一个或多个位置的氨基酸：357、394、715、719、807、844、848、857、861，即下述一个或多个氨基酸：H357、K394、R715、R719、K807、K844、N848、R857、R861；
    其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  15. 如权利要求1-14中任一项所述的工程化的Cas12i核酸酶，其中，将参比Cas12i核酸酶中与DNA-RNA双螺旋相互作用的一个或多个极性或带正电荷的氨基酸替换为疏水氨基酸包括替换为丙氨酸(A)。
  16. 如权利要求1-15中任一项所述的工程化的Cas12i核酸酶，其包含选自下组的一个或多个突变：H357A、K394A、R715A、R719A、K807A、K844A、N848A、R857A、R861A；
    优选，包含选自下组的一个或多个突变：K394A、R719A、K844A、R857A；
    其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  17. 如权利要求1-16中任一项所述的工程化的Cas12i核酸酶，其包含R719A和K844A氨基酸取代，或包含R857A和K844A氨基酸取代；其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  18. 如权利要求1～17中任一项所述的工程化的Cas12i核酸酶，其还包含一个或多个柔性区突变，所述突变增加了参比Cas12i核酸酶中的柔性区的柔性，所述柔性区选自氨基酸残基439-443或氨基酸残基925-929；
    优选，所述柔性区突变位于439和/或926位点；
    进一步优选，所述柔性区突变是L439和/或I926的突变；
    其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  19. 如权利要求18所述的工程化的Cas12i核酸酶，其中，所述一个或多个柔性区突变为：将该柔性区氨基酸替换为G、和/或在其后插入一个或两个G；
    优选，所述一个或多个柔性区突变包含I926G、L439(L+G)或L439(L+GG)；
    进一步优选，所述一个或多个柔性区突变包含L439(L+G)或L439(L+GG)；
    其中，所述氨基酸位置编号如SEQ ID NO：1所示的相应氨基酸位置所定义。
  20. 一种工程化的Cas12i核酸酶；
    所述工程化的Cas12i核酸酶包含任何以下一组或多组突变：
    (1)E563R；(2)E176R和T447R；(3)E176R和E563R；(4)K238R和E563R；(5)E176R、K238R和T447R；(6)E176R、T447R和E563R；(7)E176R、K238R和E563R；(8)E176R、K238R、T447R和E563R；(9)N164Y；(10)N164F；(11)E323R；(12)D362R；(13)Q425R；(14)N925R；(15)I926R；(16)D958R；(17)E323R和D362R；(18)E323R和Q425R；(19)E323R和I926R；(20)Q425R和I926R；(21)D362R和I926R；(22)N925R和I926R；(23)E323R、D362R和Q425R；(24)E323R、D362R和I926R；(25)E323R、Q425R和I926R；(26)D362R、N925R和I926R；(27)E323R、D362R、Q425R和I926R；(28)D362R和I926G；(29)N925R和I926G；(30)D362R、N925R和I926G；(31)I926R和L439(L+G)；(32)I926R和L439(L+GG)；(33)E323R、D362R和I926G；(34)R719A和K844A；和(35)R857A和K844A；
    优选，所述工程化的Cas12i核酸酶包含任何以下一组或多组突变：(1)E176R、K238R、T447R和E563R；(2)N164Y；(3)I926R；(4)E323R和D362R；(4)I926G；(5)I926R和L439(L+G)；(6)I926R和L439(L+GG)；和(7)D958R；
    其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  21. 一种工程化的Cas12i核酸酶，所述工程化的Cas12i核酸酶包含以下任一组突变：(1)E176R、K238R、T447R、E563R和N164Y；(2)E176R、K238R、T447R、E563R和I926R；(3)N164Y、E323R和D362R；(4)E176R、K238R、T447R、E563R、E323R和D362R；(5)N164Y和I926R；(6)E176R、K238R、T447R、E563R、N164Y和I926R；(7)E176R、K238R、T447R、E563R、N164Y、E323R和D362R；(8)E176R、K238R、T447R、E563R、N164Y、I926R、E323R和D362R；(9)E176R、K238R、T447R、E563R、 N164Y、E323R、D362R和I926G；(10)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G和L439(L+GG)；(11)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G和L439(L+G)；(12)E176R、K238R、T447R、E563R、N164Y和D958R；(13)E176R、K238R、T447R、E563R、I926R和D958R；(14)E176R、K238R、T447R、E563R、E323R、D362R和D958R；(15)N164Y、I926R和D958R；(16)N164Y、E323R、D362R和D958R；(17)E176R、K238R、T447R、E563R、N164Y、I926R和D958R；(18)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和D958R；(19)E176R、K238R、T447R、E563R、N164Y、I926R、E323R、D362R和D958R；(20)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G和D958R；(21)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G、L439(L+GG)和D958R；(22)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、I926G、L439(L+G)和D958R；(23)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和R857A；(24)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和N861A；(25)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和K807A；(26)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和N848A；(27)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和R715A；(28)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和R719A；(29)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和K394A；(30)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和H357A；(31)E176R、K238R、T447R、E563R、N164Y、E323R、D362R和K844A；(32)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、R719A和K844A；或(33)E176R、K238R、T447R、E563R、N164Y、E323R、D362R、R857A和K844A；其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  22. 如权利要求1～21中任一项所述的工程化的Cas12i核酸酶，其包含如SEQ ID NOs：2～24中任一项所示氨基酸序列的工程化Cas12i核酸酶，或与如SEQ ID NOs：2～24中任一序列所示的氨基酸序列具有至少80％同一性的氨基酸序列。
  23. 一种工程化的Cas12i效应蛋白，其包含权利要求1～22中任一项所述的工程化的Cas12i核酸酶或其功能衍生物；
    任选的，所述工程化的Cas12i核酸酶或其功能衍生物具有酶活性，或者所述工程化的Cas12i核酸酶或其功能衍生物为酶失活突变体。
  24. 如权利要求23所述的工程化的Cas12i效应蛋白，其中所述Cas12i效应蛋白能够 诱导DNA分子中的双链断裂或单链断裂。
  25. 如权利要求23所述的工程化的Cas12i效应蛋白，其中所述工程化的Cas12i核酸酶或其功能衍生物为包含以下一种或多种突变的酶失活突变体：D599A、E833A、S883A、H884A、R900A和D1019A；其中，所述氨基酸位置如SEQ ID NO：1所示的相应氨基酸位置所定义。
  26. 如权利要求23～25中任一项所述的工程化的Cas12i效应蛋白，其还包含与所述工程化的Cas12i核酸酶或其功能衍生物融合的功能结构域。
  27. 如权利要求26所述的工程化的Cas12i效应蛋白，其中所述功能结构域选自下组中的一个或多个：翻译起始结构域、转录阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域、逆转录酶结构域、报告分子结构域和核酸酶结构域。
  28. 如权利要求23-27中任一项所述的工程化的Cas12i效应蛋白，所述工程化的Cas12i效应蛋白包含：含有所述工程化的Cas12i核酸酶或其功能衍生物的N末端部分的第一多肽和含有所述工程化的Cas12i核酸酶或其功能衍生物的C末端部分的第二多肽，其中所述第一多肽和所述第二多肽能够在包含指导序列的指导RNA的存在下彼此缔合，以形成与靶核酸特异性结合的成簇规律间隔短回文重复序列(CRISPR)复合物，所述靶核酸包含与所述指导序列互补的靶序列；
    优选，所述第一多肽包含权利要求1-22中任一项所述的工程化的Cas12i核酸酶的N末端部分氨基酸残基1至X，所述第二多肽包含权利要求1-22中任一项所述的工程化的Cas12i核酸酶的氨基酸残基X+I至所述Cas12i核酸酶的C末端；
    可选的，所述第一多肽和所述第二多肽各自包含二聚化结构域；
    可选的，所述第一多肽的二聚化结构域和所述第二多肽的二聚化结构域在诱导剂存在下彼此缔合。
  29. 一种工程化的CRISPR-Cas12i系统，包括：
    (a)权利要求1-28中任一项所述的工程化的Cas12i核酸酶、或权利要求23-28中任一项所述的工程化的Cas12i效应蛋白；以及
    (b)包含与靶序列互补的指导序列的指导RNA，或编码所述指导RNA的一种或多种核酸，
    其中所述工程化的Cas12i核酸酶或工程化的Cas12i效应蛋白和所述指导RNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰；
    优选，所述指导RNA是包含所述指导序列的crRNA；
    进一步优选，所述工程化的CRISPR-Cas12i系统包括编码多个crRNA的前体指导RNA阵列(array)；
    或者，优选所述工程化的Cas12i核酸酶或工程化的Cas12i效应蛋白是主编辑器，所述指导RNA是引导编辑指导RNA(pegRNA)。
  30. 如权利要求29所述的工程化的CRISPR-Cas12i系统，其包含一种或多种编码所述工程化的Cas12i核酸酶或工程化的Cas12i效应蛋白的载体；
    优选，所述一种或多种载体选自下组：逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关的载体和单纯疱疹载体；
    进一步优选，所述一种或多种载体是腺相关病毒AAV载体；
    进一步优选，所述AAV载体还编码所述指导RNA。
  31. 一种检测样品中靶核酸的方法，包括：
    (a)使样品与权利要求29中的工程化的CRISPR-Cas12i系统以及加标签的检测核酸接触，该检测核酸为单链且不与所述指导RNA的指导序列杂交；以及
    (b)测量通过所述工程化的Cas12i核酸酶或工程化的Cas12i效应蛋白切割所述加标签的检测核酸而产生的可检测信号，从而检测所述靶核酸。
  32. 一种修饰包含靶序列的靶核酸的方法，包括使所述靶核酸与权利要求29或30所述的工程化的CRISPR-Cas12i系统接触；
    优选，所述方法在体外进行、离体进行或在体内进行；
    进一步优选，所述靶核酸存在于细胞中；
    进一步优选，所述细胞是细菌细胞、酵母细胞、哺乳动物细胞、植物细胞或动物细胞；
    进一步优选，所述靶核酸是基因组DNA；
    进一步优选，所述靶序列与疾病或病症相关；
    进一步优选，所述工程化的CRISPR-Cas12i系统包括编码多个crRNA的前体指导RNA阵列，其中每个crRNA包含不同的指导序列。
  33. 如权利要求29或30所述的工程化的CRISPR-Cas12i系统在制备治疗与个体的细胞中靶核酸相关的疾病或病症的药物中的用途；优选，所述疾病或病症选自下组：癌症、心血管疾病、遗传性疾病、自身免疫疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。
  34. 一种治疗与个体的细胞中的靶核酸相关的疾病或病症的方法，所述方法包含使用 权利要求32所述的方法来修饰所述个体的细胞中的靶核酸，从而治疗所述疾病或病症；优选，所述疾病或病症选自下组：癌症、心血管疾病、遗传性疾病、自身免疫疾病、代谢性疾病、神经退行性疾病、眼病、细菌感染和病毒感染。
  35. 一种修饰包含靶序列的靶核酸的方法，包括使所述靶核酸与权利要求29或30中所述的工程化的CRISPR-Cas12i系统接触。
  36. 包含如权利要求1-22中任一项所述的工程化的Cas12i核酸酶、如权利要求23-28中任一项所述的工程化的Cas12i效应蛋白的组合物或试剂盒。
  37. 工程化的细胞，其包含经修饰的靶核酸，其中靶核酸由如权利要求32或35所述的方法修饰。
  38. 工程化的非人类动物，其包含一种或多种如权利要求37所述的工程化的细胞。
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