Nothing Special   »   [go: up one dir, main page]

WO2021031085A1 - 一种高保真CRISPR/AsCpf1突变体的构建及其应用 - Google Patents

一种高保真CRISPR/AsCpf1突变体的构建及其应用 Download PDF

Info

Publication number
WO2021031085A1
WO2021031085A1 PCT/CN2019/101439 CN2019101439W WO2021031085A1 WO 2021031085 A1 WO2021031085 A1 WO 2021031085A1 CN 2019101439 W CN2019101439 W CN 2019101439W WO 2021031085 A1 WO2021031085 A1 WO 2021031085A1
Authority
WO
WIPO (PCT)
Prior art keywords
ascpf1
mutant
amino acid
arginine
acid sequence
Prior art date
Application number
PCT/CN2019/101439
Other languages
English (en)
French (fr)
Inventor
荣知立
林瑛
黄洪新
单琳
Original Assignee
南方医科大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南方医科大学 filed Critical 南方医科大学
Priority to EP19942232.0A priority Critical patent/EP4047087A4/en
Priority to US17/794,263 priority patent/US20230056843A1/en
Priority to PCT/CN2019/101439 priority patent/WO2021031085A1/zh
Publication of WO2021031085A1 publication Critical patent/WO2021031085A1/zh

Links

Images

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • 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
    • 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
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01037—DNA (cytosine-5-)-methyltransferase (2.1.1.37)
    • 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]
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to the field of biotechnology, in particular to the construction and application of a high-fidelity CRISPR/AsCpf1 mutant.
  • CRISPR/Cpf1 is a DNA editing technology similar to the CRISPR/Cas9 system. Like CRISPR/Cas9, it belongs to the second type of CRISPR system endonuclease. However, compared with Cas9, Cpf1 is smaller and simpler in structure, and Cpf1 also has some characteristics that Cas9 does not have. For example, Cpf1 cuts to form sticky ends, PAM region is TTTN, and has the ability to self-process CrRNA. This makes Cpf1 not only help to make up for some of the shortcomings of the CRISPR/Cas9 system, but may also have advantages over CRISPR/Cas9 in some applications.
  • CRISPR systems are widely used, especially they are gradually being used in gene therapy of diseases and improvement of degenerative diseases.
  • CRISPR systems can perform efficient gene editing on various cells, tissues, organs, etc., they also produce non-targeted cleavage at positions similar to the target site sequence, which is the so-called "off-target effect.” These off-target effects can cause some unpredictable mutations, which makes it possible to have potential risks if these nucleases are to be used in clinical practice.
  • the development of a high-fidelity CRISPR system is very critical.
  • eCas9 Cas9-HF
  • HypaCas9 evoCas9
  • Cpf1 there are few high-fidelity versions currently developed. Therefore, the patent of the present invention is based on point mutation technology, and through a series of mutation modification, a new type of Cpf1 mutant that is efficient and can reduce off-target effects is found.
  • the first objective of the present invention is to provide a new AsCpf1 mutant (AsCpf1-KA mutant) with reduced off-target effects, that is, a high-fidelity CRISPR/AsCpf1 mutant.
  • the technical solution adopted by the present invention is an AsCpf1 mutant
  • the AsCpf1 mutant is to mutate the 951st arginine in the amino acid sequence of the AsCpf1 protein, and replace it with a DNA that does not interact with the target site DNA.
  • the AsCpf1 mutant mutates the 955th arginine in the amino acid sequence of the AsCpf1 protein and replaces it with an amino acid that does not form a hydrogen bond with the target site DNA;
  • the AsCpf1 mutant is to mutate the 951st arginine and the 955th arginine of the amino acid sequence of the AsCpf1 protein, and replace them with amino acids that do not form a hydrogen bond with the target site DNA.
  • the AsCpf1 mutant is to mutate the 951st arginine and the 955th arginine of the amino acid sequence of the AsCpf1 protein, and replace them with amino acids that do not form a hydrogen bond with the target site DNA.
  • the AsCpf1 mutant is that the arginine R at position 951 in the amino acid sequence of the AsCpf1 protein is changed to lysine K, and the amino acid sequence of the AsCpf1 mutant is shown in SEQ ID NO:1;
  • the AsCpf1 mutant is that the arginine R at position 955 in the amino acid sequence of the AsCpf1 protein is changed to alanine A, and the amino acid sequence of the AsCpf1 mutant is shown in SEQ ID NO: 2;
  • the AsCpf1 mutant is that the arginine R at position 951 in the amino acid sequence of the AsCpf1 protein is changed to lysine K, and the arginine R at position 955 is changed to alanine A, the amino acid of the AsCpf1 mutant
  • the sequence is shown in SEQ ID NO: 3.
  • the AsCpf1 mutant is that the arginine R at position 951 in the amino acid sequence of the AsCpf1 protein is changed to lysine K (951R->K), and the arginine R at position 955 is changed to alanine A. (955R->A), the amino acid sequence of the AsCpf1 mutant is shown in SEQ ID NO: 3.
  • Feng Zhang team analyzed the three-dimensional structure of AsCpf1 protein (belonging to a genus commonly used in the Cpf1 family).
  • the applicant found that the R of amino acid 951 and R of 955 of AsCpf1 could form non-specific DNA with the target site DNA of the genome.
  • Heterosexual hydrogen bonding may cause AsCpf1 to form non-specific binding at positions similar to the target site sequence during gene editing, resulting in non-targeted cleavage. Therefore, the applicant made mutations in these positions to destroy the non-specific hydrogen bonding formed between the protein and the target site, so that AsCpf1 enhanced the requirement for the complementary pairing of nucleotides between the gRNA and the target site during gene editing. , Thereby reducing the off-target effect.
  • the second object of the present invention is to provide a gene encoding the aforementioned AsCpf1 mutant.
  • the technical solution adopted by the present invention is that the nucleotide sequence of the aforementioned AsCpf1 mutant encoding gene is shown in SEQ ID NO: 4.
  • the third objective of the present invention is to provide an application of the aforementioned coding gene in constructing a CRISPR/AsCpf1 gene editing system.
  • the fourth objective of the present invention is to provide a CRISPR/AsCpf1 gene editing system.
  • the technical solution adopted by the present invention is a CRISPR/AsCpf1 gene editing system containing a gene encoding the AsCpf1 protein, which is the aforementioned AsCpf1 mutant.
  • the CRISPR/AsCpf1 gene editing system also contains the U6 promoter for activating the expression of the sgRNA encoding the AsCpf1 mutant gene, the essential element CrRNA for AsCpf1 gene editing, and the eukaryotic promoter for activating the expression of the AsCpf1 mutant gene.
  • the daughter CAG, the shearing titanium sequence P2A, and the reporter gene mcherry for monitoring plasmid expression is as follows:
  • U6 promoter is used to promote the expression of sgRNA of AsCpf1 mutant gene
  • the scaffold of AsCpf1, namely CrRNA, is one of the necessary elements for AsCpf1 gene editing, and it is also an element for targeting the target target sequence;
  • the eukaryotic promoter CAG is used to initiate the expression of the AsCpf1 mutant gene
  • Red fluorescent protein mCherry fluorescent reporter gene.
  • the embodiment of the present invention selected site3 of DNMT1 (DNA methyltransferase 1) and another Match-site6.
  • the method adopted two methods: target gRNA mismatch base and target gRNA known off-target site.
  • the commonly used genotyping methods PAGE and T7E1 were used to transfect wild-type AsCpf1 plasmid (pu6-CAG-AsCpf1-mCherry wild-type AsCpf1 plasmid) and AsCpf1 mutant plasmid (pu6-CAG-AsCpf1-KA-mCherry
  • the mutant AsCpf1-KA plasmid was verified in HEK-293T (human renal epithelial cell line) and MCF7 (human breast cancer cell line).
  • the fifth object of the present invention is to provide an application of the above CRISPR/AsCpf1 gene editing system in reducing off-target effects in gene editing.
  • the gene editing off-target efficiency of the AsCpf1-KA mutant is much lower than that of the wild-type AsCpf1. Regardless of whether it is on DNMT1-Site3 where the gRNA and the target site are not completely matched, the cleavage (off-target cleavage) efficiency is much lower than that of the wild type; or on the perfectly matched Match-site6 site, PAGE and T7E1 results show that, And in different cell lines (293T or MCF7 cell lines), it can be clearly observed that the AsCpf1-KA mutant has better fidelity, which can reduce the two known off-target sites of Match-site6 to a level below the detection limit . The above results indicate that the AsCpf1-KA mutant not only has better cleavage activity on the target, but also has better specificity than wild-type AsCpf1. It is a high-fidelity CRISPR nuclease.
  • Figure 1 is a plasmid map of pU6-CAG-AsCpf1-mCherry wild-type AsCpf1.
  • Figure 2 is the plasmid map of the pU6-CAG-AsCpf1-KA-mCherry mutant AsCpf1-KA.
  • Figure 3 is a PAGE gel image of wild-type AsCpf1 (AsCpf1-WT) and AsCpf1-KA mutant (ie, AsCpf1 mutant) at the DNMT1-site3 site of the target gRNA and mismatched gRNA.
  • Figure 4 is a PAGE gel image of wild-type AsCpf1 and AsCpf1-KA mutants at the Match-site6 site at the target gRNA and 2 off-target sites, where Blank is the blank control, WT is wild-type AsCpf1, and KA is AsCpf1-KA mutant.
  • Figure 5 is the T7E1 gel image of wild-type AsCpf1 and AsCpf1-KA mutants at the Match-site6 site at the target gRNA and 2 off-target sites, where Blank is the blank control, WT is wild-type AsCpf1, and KA is AsCpf1-KA mutant.
  • Blank is the blank control
  • WT is wild-type AsCpf1
  • KA is AsCpf1-KA mutant.
  • pU6-CAG-AsCpf1-mCherry vector is digested with PmI I and BamHI to obtain the backbone vector.
  • PCR amplified fragments containing mutant bases respectively, and the PCR primer sequences are as follows:
  • AsCpf1-Pml I-F 5'-ACCAGCGACAAGTTCTTTTTCCACGTGCCTATCA-3'; (SEQ ID NO: 6)
  • AsCpf1-KA-R 5'-CACAGACCAGGCCTGAGCGGCCGCCACCTTCTCCTTCTCCCTGTTG-3'; (SEQ ID NO: 7)
  • AsCpf1-KA-F 5'-GAAGGAGAAGGTGGCGGCCGCTCAGGCCTGGTCTGTGGTGGGC-3'; (SEQ ID NO: 8)
  • AsCpf1-BamH I-R 5'-AAGCGTAATCTGGAACATCGTATGGGTAGGATCC-3'; (SEQ ID NO: 9)
  • PCR was performed with NEB Q5 enzyme (50 ⁇ l system is as follows: 5 ⁇ reaction buffer: 10 ⁇ l; 5 ⁇ enchance GC buffer: 10 ⁇ l; dNTP Mix 2.5 ⁇ m each: 4 ⁇ l; F+R: 2 +2 ⁇ l; Template: 2ng DNA; water: up to 50 ⁇ l. Reaction conditions: 98°C15s, 35cycle (98°C10s, 58°C30s, 72°C1kb/s), 72°C10min, 4°Chold), run the gel, The single product obtained was purified using PCR purification kit.
  • wild-type AsCpf1 can cleave some even incompletely matched gRNAs, especially 1 , 2, 8, 9, 19, 20, 21, 22, 23 and other positions. In other words, wild-type AsCpf1 can tolerate some base mismatches of gRNA, that is, the specificity is average.
  • gRNA for the DNMT1-site3 site to verify the reference, including the perfectly matched on-target site (ON), mismatch 1 (mm1), mismatch 8 (mm8), and mismatch 9 (mm9), mismatch 19 (mm19), mismatch 20 (mm20) and other positions of gRNA, the specific sequence is as follows:
  • AsCpf1-gRNA-DNMT1-3-ON CTGATGGTCCATGTCTGTTACTC; (SEQ ID NO: 10)
  • AsCpf1-gRNA-DNMT1-3-mm1 G TGATGGTCCATGTCTGTTACTC (underlined is the position of mismatch); (SEQ ID NO: 11)
  • AsCpf1-gRNA-DNMT1-3-mm8 CTGATGG A CCATGTCTGTTACTC (underlined is the position of mismatch); (SEQ ID NO: 12)
  • AsCpf1-gRNA-DNMT1-3-mm9 CTGATGGT G CATGTCTGTTACTC (underline is the position of mismatch); (SEQ ID NO: 13)
  • AsCpf1-gRNA-DNMT1-3-mm19 CTGATGGTCCATGTCTGT A ACTC (underlined is the position of mismatch); (SEQ ID NO: 14)
  • AsCpf1-gRNA-DNMT1-3-mm20 CTGATGGTCCATGTCTGTT T CTC (the position of mismatch is underlined); (SEQ ID NO: 15)
  • pU6-CAG-AsCpf1-mCherry-ON that is, pU6-CAG-AsCpf1-mCherry is connected to AsCpf1-gRNA-DNMT1-3-ON, and so on
  • pU6-CAG-AsCpf1-KA-mCherry-ON pU6- CAG-AsCpf1-KA-mCherry-mm1, pU6-CAG-AsCpf1-KA-mCherry-mm8, pU6
  • the 12 expression plasmids constructed above were transferred into HEK293T with the transfection reagent PEI. After 48 hours, the cells were digested, the genomic DNA was extracted by SDS lysis method, and the genomic DNA was used as a template for PCR amplification.
  • the primers are as follows:
  • DNMT1-3-PAGE-F 5'-CAAGTGCTTAGAGCAGGCGT-3'; (SEQ ID NO: 16)
  • DNMT1-3-PAGE-R 5'-GTGACGGGAGGGCAGAACTA-3'; (SEQ ID NO: 17)
  • the PCR reaction system is as follows:
  • gDNA 50ng; F+R primer: 0.5 ⁇ l; 2 ⁇ PCR mix: 5 ⁇ l; water: up to 10 ⁇ l.
  • Reaction conditions 95°C for 5min; 40cycle (95°C for 30s, 58°C for 30s, 72°C for 20s), 72°C for 10min, 95°C for 5min, and then naturally cooled to room temperature.
  • pU6-pCAG-AsCpf1-mCherry and pU6-pCAG-AsCpf1-KA-mCherry were used as vectors, respectively, and a 9452bp vector was obtained by digestion with BaeI.
  • the target gRNA-oligo-F and R were synthesized and annealed to obtain site6-ON.
  • the two expression plasmids constructed above were transformed into HEK293T or MCF7 with the transfection reagent PEI. After 48 hours, the cells were digested, the genomic DNA was extracted by SDS lysis method, and PCR amplification was performed using the genomic DNA as a template, and then T7E1 and PAGE were used Glue is used for editing efficiency and specificity analysis.
  • the primers used are as follows:
  • Site6-ON-F 5'-CCACATCCTCACCACCTGTT-3'; (SEQ ID NO: 21)
  • Site6-ON-R 5'-CCCACAGCCATCCAGCTC-3'; (SEQ ID NO: 22)
  • Site6-OT1-PAGE-F 5'-ACACTACGATGGTCCCTGGTGC-3'; (SEQ ID NO: 23)
  • Site6-OT1-PAGE-R 5'-TGGATGCTGGATGGCGTCACAT-3'; (SEQ ID NO: 24)
  • Site6-OT1-T7E1-F 5'-AGCCAATATTATTACATTGCCGTT-3'; (SEQ ID NO: 25)
  • Site6-OT1-T7E1-R 5'-TGGCGTCACATTAGTGCCAT-3'; (SEQ ID NO: 26)
  • Site6-OT2-PAGE-F 5'-GACTTGGCTAGCTTGGGGAC-3'; (SEQ ID NO: 27)
  • Site6-OT2-PAGE-R 5'-GCTGTGAGAAACCCCATGTT-3'; (SEQ ID NO: 28)
  • Site6-OT2-T7E1-F 5'-GACAGTTCAGACCCTTGGGG-3'; (SEQ ID NO: 29)
  • Site6-OT2-T7E1-R 5'-TGCTGTGAGAAACCCCATGTT-3'; (SEQ ID NO: 30)
  • the T7E1 identification method is as follows:
  • gDNA 50ng
  • F+R primer 2 ⁇ l
  • 2 ⁇ PCR mix 15 ⁇ l
  • water up to 30 ⁇ l
  • the PAGE gel identification method is as follows:
  • gDNA 50ng
  • PAGE-F+R primer 0.5 ⁇ l
  • 2 ⁇ PCR mix 5 ⁇ l
  • water up to 10 ⁇ l
  • Reaction conditions 95°C for 5min; 40cycle (95°C for 30s, 58°C for 30s, 72°C for 20s), 72°C for 10min, 95°C for 5min, and then naturally cooled to room temperature;
  • PAGE gel and T7E1 show editing efficiency and specificity shown in Figure 4 and Figure 5.
  • the gene editing off-target efficiency of mutant AsCpf1-KA was much lower than that of wild-type AsCpf1.
  • the cleavage (off-target cleavage) efficiency is much lower than that of the wild type; or on the perfectly matched Match-site6 site, the results of PAGE and T7E1 show that, And in different cell lines (293T or MCF7 cell lines), it can be clearly observed that the fidelity of the mutant AsCpf1-KA is better.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Virology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

提供一种高保真CRISPR/AsCpf1突变体的构建及其应用，所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸和/或第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸，其氨基酸序列如SEQ ID NO：1-3所示，所述的AsCpf1突变体的编码基因，其核苷酸序列如SEQ ID NO：4所示；还提供所述的编码基因在构建CRISPR/AsCpf1基因编辑系统中的应用，一种CRISPR/AsCpf1基因编辑系统，含有编码AsCpf1蛋白的基因，所述AsCpf1蛋白为上述所述的AsCpf1突变体；还提供了CRISPR/AsCpf1基因编辑系统在基因编辑中降低脱靶效应中的应用；所述构建的新型AsCpf1突变体既保留野生型AsCpf1的基因编辑效率，同时比野生型AsCpf1具有较高的特异性。

Description

一种高保真CRISPR/AsCpf1突变体的构建及其应用 技术领域
本发明涉及生物技术领域，具体涉及一种高保真CRISPR/AsCpf1突变体的构建及其应用。
背景技术
CRISPR/Cpf1是与CRISPR/Cas9系统相似的DNA编辑技术，它与CRISPR/Cas9一样，属于二类CRISPR系统核酸内切酶。但是，和Cas9相比，Cpf1更小，结构也更简单，而且Cpf1还具有Cas9没有的一些特性，如Cpf1切割形成的是粘性末端，PAM区为TTTN，且自身具有自加工CrRNA能力等特点，使得Cpf1不但可以帮助弥补CRISPR/Cas9系统的一些缺陷，而且可能在某些方面的应用上，比CRISPR/Cas9更有优势。目前CRISPR系统应用广泛，特别是它们逐渐被应用于疾病的基因治疗及改善退行性病变等方面。虽然CRISPR系统可以对各种细胞、组织、器官等进行高效基因编辑，但它们也会在一些与靶位点序列相似的位置产生非靶向的切割，这就是所谓的“脱靶效应”。这些脱靶效应会引起一些不可预测的突变，这就使得，如果要将这些核酸酶应用于临床，则可能存在潜在风险。基于此，开发高保真的CRISPR系统就显得非常关键。目前对于高保真Cas9已经开发了好几个版本，例如eCas9，Cas9-HF，HypaCas9，evoCas9等等。而对于Cpf1来说，其目前开发的高保真版本则很少。所以，本发明专利基于点突变技术，通过一系列突变改造，找到了一个高效，且可以降低脱靶效应的新型Cpf1突变体。
发明内容
本发明的第一个目的是提供一种新的，具有降低脱靶效应的AsCpf1突变体(AsCpf1-KA突变体)，即高保真的CRISPR/AsCpf1突变体。
为了达到上述目的，本发明所采用的技术方案为，一种AsCpf1突变体，所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸；
或所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸；
或所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸和第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸。
进一步地，所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸和第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸。
进一步地，所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第951位的精氨酸R变为赖氨酸K，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：1所示；
或所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第955位的精氨酸R变为丙氨酸A，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：2所示；
或所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第951位的精氨酸R变为赖氨酸K，以及第955位的精氨酸R变为丙氨酸A，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：3所示。
进一步地，所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第951位的精氨酸R变为赖氨酸K(951R->K)，以及第955位的精氨酸R变为丙氨酸A(955R->A)，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：3所示。
2016年Feng Zhang小组解析了AsCpf1蛋白(属于Cpf1家族中常用一个菌属)的三维结构，申请人通过研究分析，发现AsCpf1的氨基酸951的R和955的R可与基因组的靶位点DNA形成非特异性的氢键结合，这可能导致AsCpf1在进行基因编辑时，会在一些与靶位点序列相似的位置形成非特异性结合，从而产生非靶向切割。因此申请人对这几个位置进行突变，破坏蛋白与靶位点之间形成的非特异性的氢键结合，使得AsCpf1在基因编辑时对gRNA与靶位点之间核苷酸的互补配对要求加强，从而降低脱靶效应。
本发明的第二个目的是提供一种上述AsCpf1突变体的编码基因。
为了达到上述目的，本发明所采用的技术方案为，上述所述的AsCpf1突变体的编码基因，其核苷酸序列如SEQ ID NO：4所示。
本发明的第三个目的是提供一种上述所述的编码基因在构建CRISPR/AsCpf1基因编辑系统中的应用。本发明的第四个目的是提供一种CRISPR/AsCpf1基因编辑系统。
为了达到上述目的，本发明所采用的技术方案为，一种CRISPR/AsCpf1基因编辑系统，含有编码AsCpf1蛋白的基因，所述AsCpf1蛋白为上述所述的AsCpf1突变体。
进一步地，所述CRISPR/AsCpf1基因编辑系统还含有用于启动编码AsCpf1突变体基因的sgRNA表达的U6启动子、AsCpf1基因编辑的必须元件CrRNA、用于启动编码AsCpf1突变体基因表达的真核启动子CAG、剪切钛序列P2A，以及监测质粒表达的报告基因mcherry。具体效用如下：
1、U6启动子，作用为启动AsCpf1突变体基因的sgRNA的表达；
2、AsCpf1的scaffold，即CrRNA，AsCpf1基因编辑的必须元件之一，同时也是导向目标靶序列的元件；
3、真核启动子CAG，用来启动AsCpf1突变体基因的表达；
4、剪切钛序列P2A，蛋白连接柔性序列；
5、红色荧光蛋白mCherry，荧光报告基因。
作为特异性验证，本发明实施例选取了DNMT1(DNA甲基转移酶1)的site3和另一个Match-site6。方法上采取了目标gRNA错配碱基和目标gRNA已知脱靶位点两种方法。而技术方面采用了常用的基因型分析方法PAGE和T7E1，通过转染野生型AsCpf1质粒(pu6-CAG-AsCpf1-mCherry野生型AsCpf1质粒)与AsCpf1突变体质粒(pu6-CAG-AsCpf1-KA-mCherry突变体AsCpf1-KA质粒)，在HEK-293T(人肾上皮细胞系)与MCF7(人乳腺癌细胞系)中进行验证。结果证明，不管是在错配碱基或是在已知的脱靶位点上，且在不同细胞株293T或MCF7中，突变的AsCpf1突变体(AsCpf1-KA)的特异性都要比野生型AsCpf1好。
本发明的第五个目的是提供一种上述CRISPR/AsCpf1基因编辑系统在基因编辑中降低脱靶效应中的应用。
与现有技术相比，本发明的优势在于：
本发明提供的AsCpf1-KA突变体(即AsCpf1突变体)的基因编辑脱靶效率要远低于野生型的AsCpf1。不管是在gRNA与靶位点不完全匹配的DNMT1-Site3上，其切割(脱靶的切割)效率要远低于野生型；还是在完全匹配的Match-site6位点上，PAGE和T7E1结果显示，以及在不同的细胞系(293T或MCF7细胞系)中，都可以明显观察到AsCpf1-KA突变体保真性更好，它能降低Match-site6的2个已知脱靶位点到检测限以下的水平。以上结果表明，AsCpf1-KA突变体不但具有较好的在靶切割活性，而且其特异性比野生型AsCpf1更好，是一种高保真的CRISPR核酸酶。
附图说明
图1为pU6-CAG-AsCpf1-mCherry野生型AsCpf1质粒图谱。
图2为pU6-CAG-AsCpf1-KA-mCherry突变体AsCpf1-KA质粒图谱。
图3为野生型AsCpf1(AsCpf1-WT)和AsCpf1-KA突变体(即AsCpf1突变体)在DNMT1-site3位点的在靶gRNA与错配gRNA的PAGE胶图。
图4为野生型AsCpf1和AsCpf1-KA突变体在Match-site6位点的在靶gRNA与2个脱靶位点的PAGE胶图，其中Blank为空白对照，WT为野生型AsCpf1，KA为AsCpf1-KA突变体。
图5为野生型AsCpf1和AsCpf1-KA突变体在Match-site6位点的在靶gRNA与2个脱靶位点的T7E1胶图，其中Blank为空白对照，WT为野生型AsCpf1，KA为AsCpf1-KA突变体。(左：细胞系HEK293T，右：细胞系MCF7，黑色箭头表示脱靶切割情况)
具体实施方式
下面结合具体实施例对本发明做进一步详细的说明，但本发明并不限于以下实施例。除非特别说明，下面实施例中所用的技术均为本领域内的技术人员已知的常规技术，所使用的仪器设备和试剂等，均为本领域内的技术人员可以通过公共途径如商购等获得的。
实施例1 重组表达质粒构建
pU6-CAG-AsCpf1-mCherry序列如SEQ ID NO：5所示。
以野生型pU6-CAG-AsCpf1-mCherry(图1)为载体(其核苷酸序列如SEQ ID NO：5所示)，利用Gibson Assembly原理技术，设计相对应突变引物，进行PCR，最后连接得到pU6-CAG-AsCpf1-KA-mCherry(图2)。具体如下：
先将pU6-CAG-AsCpf1-mCherry载体进行PmI I和BamHI酶切得到骨架载体。以pU6-CAG-AsCpf1-mCherry为模板，PCR分别扩增得到含突变碱基的片段，PCR引物序列如下：
AsCpf1-Pml I-F：5'-ACCAGCGACAAGTTCTTTTTCCACGTGCCTATCA-3'；(SEQ ID NO：6)
AsCpf1-KA-R：5'-CACAGACCAGGCCTGAGCGGCCGCCACCTTCTCCTTCTCCCTGTTG-3'；(SEQ ID NO：7)
AsCpf1-KA-F：5'-GAAGGAGAAGGTGGCGGCCGCTCAGGCCTGGTCTGTGGTGGGC-3'；(SEQ ID NO：8)
AsCpf1-BamH I-R：5'-AAGCGTAATCTGGAACATCGTATGGGTAGGATCC-3'；(SEQ ID NO：9)
以质粒pU6-CAG-AsCpf1-mCherry为模板，NEB Q5酶进行PCR(50μl体系如下：5×reaction buffer：10μl；5×enchance GC buffer：10μl；dNTP Mix 2.5μm each：4μl；F+R：2+2μl；Template：2ng DNA；水：up to 50μl。反应条件：98℃15s，35cycle(98℃10s，58℃30s，72℃1kb/s)，72℃10min，4℃hold)，跑胶，将得到的单一产物，应用PCR纯化试剂盒进行纯化。
其中AsCpf1-Pml I-F与AsCpf1-KA-R进行PCR，其产物命名为PCR-片段1；
AsCpf1-KA-F与AsCpf1-Bam H I-R进行PCR，其产物命名为PCR-片段2。
利用Gibson Assembly试剂盒将PCR片段1，PCR片段2以及使用限制性内切酶PmI I和BamHI对pU6-CAG-AsCpf1-mCherry载体进行双酶切后得到的骨架载体行3片段Gibson Assembly反应即可得到目标质粒pU6-CAG-AsCpf1-KA-mCherry(图2)，该质粒只是对pU6-CAG-AsCpf1-mCherry载体中的AsCpf1蛋白的氨基酸序列的第951位的精氨酸R变为赖氨酸K(951R->K)，以及第955位的精氨酸R变为丙氨酸A(955R->A)，突变后的AsCpf1突变体的氨基酸序列如SEQ ID NO：3所示。
实施例2 特异性验证
为了验证得到的突变体AsCpf1-KA(即AsCpf1突变体)保真性比野生型AsCpf1好，设计以下实验进行特异性验证：
(1)文献报道(BP Kleinstiver,et al.Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells,Nartue.2016)，野生型AsCpf1对一些即使不完全匹配的gRNA也能进行切割，特别是1，2，8，9，19，20，21，22，23等位置。也就是说野生型AsCpf1能容忍一些gRNA的碱基错配，即特异性一般。所以，我们参考文献设计了针对DNMT1-site3这个位点的gRNA进行了验证，其中包括完全匹配的在靶位点(ON)，和错配1(mm1)，错配8(mm8)，错配9(mm9)，错配19(mm19)，错配20(mm20)等位置的gRNA，具体序列如下：
AsCpf1-gRNA-DNMT1-3-ON：CTGATGGTCCATGTCTGTTACTC；(SEQ ID NO：10)
AsCpf1-gRNA-DNMT1-3-mm1： GTGATGGTCCATGTCTGTTACTC(下划线为错配位置)；(SEQ ID NO：11)
AsCpf1-gRNA-DNMT1-3-mm8：CTGATGG ACCATGTCTGTTACTC(下划线为错配位置)；(SEQ ID NO：12)
AsCpf1-gRNA-DNMT1-3-mm9：CTGATGGT GCATGTCTGTTACTC(下划线为错配位置)；(SEQ ID NO：13)
AsCpf1-gRNA-DNMT1-3-mm19：CTGATGGTCCATGTCTGT AACTC(下划线为错配位置)；(SEQ ID NO：14)
AsCpf1-gRNA-DNMT1-3-mm20：CTGATGGTCCATGTCTGTT TCTC(下划线为错配位置)；(SEQ ID NO：15)
分别以pU6-pCAG-AsCpf1-mCherry和pU6-pCAG-AsCpf1-KA-mCherry为载体，用BaeI酶切得到9452bp的载体，合成相对应的gRNA-oligo-F和R，并将其退火得到gRNA的DNA序列，利用T4ligase试剂盒将载体与完全匹配或带错配的gRNA相连，即可得到质粒：
pU6-CAG-AsCpf1-mCherry-ON(即pU6-CAG-AsCpf1-mCherry与AsCpf1-gRNA-DNMT1-3-ON连接，以下以此类推)，pU6-CAG-AsCpf1-mCherry-mm1，pU6-CAG-AsCpf1-mCherry-mm8，pU6-CAG-AsCpf1-mCherry-mm9，pU6-CAG-AsCpf1-mCherry-mm19，pU6-CAG-AsCpf1-mCherry-mm20；pU6-CAG-AsCpf1-KA-mCherry-ON，pU6-CAG-AsCpf1-KA-mCherry-mm1，pU6-CAG-AsCpf1-KA-mCherry-mm8，pU6-CAG-AsCpf1-KA-mCherry-mm9，pU6-CAG-AsCpf1-KA-mCherry-mm19，pU6-CAG-AsCpf1-KA-mCherry-mm20，
将上述构建好的12个表达质粒用转染试剂PEI转进HEK293T，48小时后，消化细胞，用SDS裂解法提取基因组DNA，以基因组DNA为模板进行PCR扩增，引物如下：
DNMT1-3-PAGE-F：5'-CAAGTGCTTAGAGCAGGCGT-3'；(SEQ ID NO：16)
DNMT1-3-PAGE-R：5'-GTGACGGGAGGGCAGAACTA-3'；(SEQ ID NO：17)
PCR反应体系如下：
gDNA：50ng；F+R引物：0.5μl；2×PCR mix：5μl；水：up to 10μl。
反应条件：95℃5min；40cycle(95℃30s，58℃30s，72℃20s)，72℃10min，95℃5min，然后自然降温至室温。
1)取PCR产物2ul加样，恒流12m Ah，跑PAGE胶，当溴酚蓝条带移动到距凝胶前沿约1cm时，停止电泳；
2)到达时间点后，将PAGE胶从玻璃板轻轻剥出，放入含GelRed的溶液中浸泡3min，然后于紫外下拍照并观察；
3)PAGE胶进行基因型鉴定，观察编辑效率和特异性情况，结果见图3。结果显示，野生型AsCpf1用错配的gRNA(1，8，9，19，20位)也能检测出明显的切割效果(出现明显的杂带，特别是20位错配其基本与不错配的gRNA显示出一样编辑效率28％)；而改造的AsCpf1-KA用错配的gRNA(1，8，9，19，20位)检测出的切割效率明显比野生型低，基本都保持在10％以下，但同时也保持了几乎与野生型(28％)一样的效率，达24％编辑效率。
(2)另外，有文献报道野生型AsCpf1在Match-site6上存在着明显的脱靶位点，于是申请人挑出了其中2个进行验证。在靶的gRNA和2个脱靶gRNA位置及序列如下：
Chr3：Match-site6-ON：GGGTGATCAGACCCAACAGCAGG；(SEQ ID NO：18)
Chr2：Match-site6-OT1：GGGTGATCAGACCCAACA CCAGG(下划线为脱靶位置)；(SEQ ID NO：19)
Chr8：Match-site6-OT2：GGGTGATCAGACCCAACA CCAGG(下划线为脱靶位置)；(SEQ ID NO：20)
同样，分别以pU6-pCAG-AsCpf1-mCherry和pU6-pCAG-AsCpf1-KA-mCherry为载体，用BaeI酶切得到9452bp的载体，合成在靶的gRNA-oligo-F和R，退火得到site6-ON-gRNA的DNA序列，利用T4ligase试剂盒将载体与gRNA的DNA序列相连，即可得到质粒：
pU6-CAG-AsCpf1-mCherry-site6-ON
pU6-CAG-AsCpf1-KA-mCherry-site6-ON
将上述构建好的2个表达质粒用转染试剂PEI转进HEK293T或MCF7，48小时后，消化细胞，用SDS裂解法提取基因组DNA，以基因组DNA为模板进行PCR扩增，然后应用T7E1和PAGE胶进行编辑效率及特异性分析，其中所用引物如下：
Site6-ON-F：5'-CCACATCCTCACCACCTGTT-3'；(SEQ ID NO：21)
Site6-ON-R：5'-CCCACAGCCATCCAGCTC-3'；(SEQ ID NO：22)
Site6-OT1-PAGE-F：5'-ACACTACGATGGTCCCTGGTGC-3'；(SEQ ID NO：23)
Site6-OT1-PAGE-R：5'-TGGATGCTGGATGGCGTCACAT-3'；(SEQ ID NO：24)
Site6-OT1-T7E1-F：5'-AGCCAATATTATTACATTGCCGTT-3'；(SEQ ID NO：25)
Site6-OT1-T7E1-R：5'-TGGCGTCACATTAGTGCCAT-3'；(SEQ ID NO：26)
Site6-OT2-PAGE-F：5'-GACTTGGCTAGCTTGGGGAC-3'；(SEQ ID NO：27)
Site6-OT2-PAGE-R：5'-GCTGTGAGAAACCCCATGTT-3'；(SEQ ID NO：28)
Site6-OT2-T7E1-F：5'-GACAGTTCAGACCCTTGGGG-3'；(SEQ ID NO：29)
Site6-OT2-T7E1-R：5'-TGCTGTGAGAAACCCCATGTT-3'；(SEQ ID NO：30)
T7E1鉴定方法具体如下：
1)提取细胞基因组DNA；
2)取50ng gDNA为模板行PCR，体系如下：gDNA：50ng；F+R引物：2μl；2×PCR mix：15μl；水：up to 30μl；
反应条件：95℃5min；38cycle(95℃30s，58℃30s，72℃20s)，72℃10min，4℃Hold；
3)纯化PCR产物，之后取300ng DNA行退火步骤：
纯化DNA：300ng；NEB buffer 2：2ul；水：up to 20μl；
反应条件：95℃5min，接着自然降温至室温，然后加入0.3ul T7E1内切酶37℃反应4h，之后跑胶于紫外下拍照观察。
PAGE胶鉴定方法具体如下：
1)提取细胞基因组DNA；
2)取50ng gDNA为模板行PCR，体系如下：gDNA：50ng；PAGE-F+R引物：0.5μl； 2×PCR mix：5μl；水：up to 10μl；
反应条件：95℃5min；40cycle(95℃30s，58℃30s，72℃20s)，72℃10min，95℃5min，然后自然降温至室温；
3)取PCR产物2ul加样，恒流12m Ah，跑PAGE胶，当溴酚蓝条带移动到距凝胶前沿约1cm时，停止电泳；
4)到达时间点后，将PAGE胶从玻璃板轻轻剥出，放入含GelRed的溶液中浸泡3min，然后于紫外下拍照并观察。
PAGE胶和T7E1显示编辑效率和特异性情况见图4和图5。结果，与预期一致，突变体AsCpf1-KA的基因编辑脱靶效率要远低于野生型的AsCpf1。不管是在gRNA与靶位点不完全匹配的DNMT1-site3上，其切割(脱靶的切割)效率要远低于野生型；还是在完全匹配的Match-site6位点上，PAGE和T7E1结果显示，以及在不同的细胞系(293T或MCF7细胞系)中，都可以明显观察到突变体AsCpf1-KA保真性更好，它能降低Match-site6的2个已知脱靶点到检测限以下的水平，黑色箭头所示。所有这些结果都说明了突变体AsCpf1-KA不但具有较好的在靶切割活性，而且其特异性比野生型AsCpf1更好，是一种高保真的CRISPR核酸酶。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种AsCpf1突变体，其特征在于，所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸；
    或所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸；
    或所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸和第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸。
  2. 根据权利要求1所述的AsCpf1突变体，其特征在于，所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸和第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸。
  3. 根据权利要求1所述的AsCpf1突变体，其特征在于，所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第951位的精氨酸R变为赖氨酸K，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：1所示；
    或所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第955位的精氨酸R变为丙氨酸A，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：2所示；
    或所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第951位的精氨酸R变为赖氨酸K，以及第955位的精氨酸R变为丙氨酸A，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：3所示。
  4. 根据权利要求3所述的AsCpf1突变体，其特征在于，所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第951位的精氨酸R变为赖氨酸K，以及第955位的精氨酸R变为丙氨酸A，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：3所示。
  5. 一种权利要求1-4任一所述的AsCpf1突变体的编码基因。
  6. 根据权利要求5所述的编码基因，其特征在于，其核苷酸序列如SEQ ID NO：4所示。
  7. 权利要求5或6所述的编码基因在构建CRISPR/AsCpf1基因编辑系统中的应用。
  8. 一种CRISPR/AsCpf1基因编辑系统，含有编码AsCpf1蛋白的基因，其特征在于，所述AsCpf1蛋白为权利要求1-4任一所述的AsCpf1突变体。
  9. 根据权利要求8所述的CRISPR/AsCpf1基因编辑系统，其特征在于，所述CRISPR/AsCpf1基因编辑系统还含有用于启动编码AsCpf1突变体基因的sgRNA表达的U6启动子、AsCpf1基因编辑的必须元件CrRNA、用于启动编码AsCpf1突变体基因表达的真核启动子CAG和剪切钛序列P2A。
  10. 权利要求8或9所述的CRISPR/AsCpf1基因编辑系统在基因编辑中降低脱靶效应中 的应用。
PCT/CN2019/101439 2019-08-19 2019-08-19 一种高保真CRISPR/AsCpf1突变体的构建及其应用 WO2021031085A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP19942232.0A EP4047087A4 (en) 2019-08-19 2019-08-19 Construction of high-fidelity crispr/ascpf1 mutant and application thereof
US17/794,263 US20230056843A1 (en) 2019-08-19 2019-08-19 Construction of high-fidelity crispr/ascpf1 mutant and uses thereof
PCT/CN2019/101439 WO2021031085A1 (zh) 2019-08-19 2019-08-19 一种高保真CRISPR/AsCpf1突变体的构建及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/101439 WO2021031085A1 (zh) 2019-08-19 2019-08-19 一种高保真CRISPR/AsCpf1突变体的构建及其应用

Publications (1)

Publication Number Publication Date
WO2021031085A1 true WO2021031085A1 (zh) 2021-02-25

Family

ID=74660150

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/101439 WO2021031085A1 (zh) 2019-08-19 2019-08-19 一种高保真CRISPR/AsCpf1突变体的构建及其应用

Country Status (3)

Country Link
US (1) US20230056843A1 (zh)
EP (1) EP4047087A4 (zh)
WO (1) WO2021031085A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115960866A (zh) * 2023-01-05 2023-04-14 河南农业大学 一种Mb2Cas12a-RRVRR突变体蛋白及其制备方法和应用
WO2023232109A1 (zh) * 2022-06-01 2023-12-07 中国科学院遗传与发育生物学研究所 新的crispr基因编辑系统
US12201699B2 (en) 2014-10-10 2025-01-21 Editas Medicine, Inc. Compositions and methods for promoting homology directed repair

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116751763B (zh) * 2023-05-08 2024-02-13 珠海舒桐医疗科技有限公司 一种Cpf1蛋白、V型基因编辑系统及应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017127807A1 (en) * 2016-01-22 2017-07-27 The Broad Institute Inc. Crystal structure of crispr cpf1
CN107312761A (zh) * 2017-07-18 2017-11-03 江苏溥博生物科技有限公司 一种AsCpf1突变体蛋白、编码基因、重组表达载体及其制备方法与应用
CN107488649A (zh) * 2017-08-25 2017-12-19 南方医科大学 一种Cpf1和p300核心结构域的融合蛋白、相应的DNA靶向激活系统和应用
WO2019118516A1 (en) * 2017-12-11 2019-06-20 Editas Medicine, Inc. Cpf1-related methods and compositions for gene editing

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9790490B2 (en) * 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
US10648020B2 (en) * 2015-06-18 2020-05-12 The Broad Institute, Inc. CRISPR enzymes and systems
WO2017106657A1 (en) * 2015-12-18 2017-06-22 The Broad Institute Inc. Novel crispr enzymes and systems
US11352647B2 (en) * 2016-08-17 2022-06-07 The Broad Institute, Inc. Crispr enzymes and systems
US11866697B2 (en) * 2017-05-18 2024-01-09 The Broad Institute, Inc. Systems, methods, and compositions for targeted nucleic acid editing
US20200181623A1 (en) * 2017-05-18 2020-06-11 The Broad Institute, Inc. Systems, methods, and compositions for targeted nucleic acid editing
EP3645054A4 (en) * 2017-06-26 2021-03-31 The Broad Institute, Inc. CRISPR / CAS-ADENINE DESAMINASE COMPOSITIONS, TARGETED NUCLEIC ACID EDITING SYSTEMS AND METHODS

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017127807A1 (en) * 2016-01-22 2017-07-27 The Broad Institute Inc. Crystal structure of crispr cpf1
CN107312761A (zh) * 2017-07-18 2017-11-03 江苏溥博生物科技有限公司 一种AsCpf1突变体蛋白、编码基因、重组表达载体及其制备方法与应用
CN107488649A (zh) * 2017-08-25 2017-12-19 南方医科大学 一种Cpf1和p300核心结构域的融合蛋白、相应的DNA靶向激活系统和应用
WO2019118516A1 (en) * 2017-12-11 2019-06-20 Editas Medicine, Inc. Cpf1-related methods and compositions for gene editing

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BP KLEINSTIVER ET AL.: "Genome-wide specificities of CRISPR-Cas Cpfl nucleases in human cells", NARTUE, 2016
YAMANO, T., ET AL.: "Crystal structure of Cpf1 in complex with guide RNA and target DNA.", CELL., vol. 165, no. 4, 5 May 2016 (2016-05-05), XP029530759, DOI: 20200507180415X *
YAMANO, T., ET AL.: "Crystal structure of Cpf1 in complex with guide RNA and target DNA.", CELL., vol. 165, no. 4, 5 May 2016 (2016-05-05), XP029530759, DOI: 20200507180428Y *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12201699B2 (en) 2014-10-10 2025-01-21 Editas Medicine, Inc. Compositions and methods for promoting homology directed repair
WO2023232109A1 (zh) * 2022-06-01 2023-12-07 中国科学院遗传与发育生物学研究所 新的crispr基因编辑系统
CN115960866A (zh) * 2023-01-05 2023-04-14 河南农业大学 一种Mb2Cas12a-RRVRR突变体蛋白及其制备方法和应用

Also Published As

Publication number Publication date
EP4047087A1 (en) 2022-08-24
US20230056843A1 (en) 2023-02-23
EP4047087A4 (en) 2023-08-23

Similar Documents

Publication Publication Date Title
WO2021031085A1 (zh) 一种高保真CRISPR/AsCpf1突变体的构建及其应用
Lee et al. Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome-and transcriptome-wide off-target effects
Bai et al. CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish
US11104897B2 (en) Compositions and methods for the treatment of nucleotide repeat expansion disorders
US10011850B2 (en) Using RNA-guided FokI Nucleases (RFNs) to increase specificity for RNA-Guided Genome Editing
KR101886381B1 (ko) 불활성화된 표적 특이적 뉴클레아제를 이용한 표적 dna의 분리 방법
EP3074515B1 (en) Somatic haploid human cell line
JP2023529151A (ja) プログラム可能なヌクレアーゼ及び使用方法
WO2016082135A1 (zh) 一种利用定点切割系统对猪h11位点定点插入的方法
US20220340936A1 (en) Programmable polynucleotide editors for enhanced homologous recombination
WO2019227640A1 (zh) 利用碱基编辑修复fbn1t7498c突变的试剂和方法
EP3940078A1 (en) Off-target single nucleotide variants caused by single-base editing and high-specificity off-target-free single-base gene editing tool
CN110300802A (zh) 用于动物胚胎碱基编辑的组合物和碱基编辑方法
WO2020087631A1 (zh) 基于C2c1核酸酶的基因组编辑系统和方法
CN113249362B (zh) 经改造的胞嘧啶碱基编辑器及其应用
CN116286905B (zh) 牛源化CRISPR/boCas9基因编辑系统、方法及应用
WO2023016021A1 (zh) 一种碱基编辑工具及其构建方法
CN113564145B (zh) 用于胞嘧啶碱基编辑的融合蛋白及其应用
CN116004716A (zh) 一种复制型dCas9-FokI系统进行高效基因编辑的方法
CN118147363A (zh) 新型CRISPR-Cas13n酶和系统
CN112391410B (zh) 一种sgRNA及其在修复内含子异常剪接中的应用
CN116144629A (zh) Cas9蛋白、含有Cas9蛋白的基因编辑系统及应用
CN105713885A (zh) 特异识别并修复β地中海贫血症beta-globin基因的嵌合核酸酶
Xie et al. Undetectable off-target effects induced by FokI catalytic domain in mouse embryos
WO2021155607A1 (zh) 经改造的胞嘧啶碱基编辑器及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19942232

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019942232

Country of ref document: EP

Effective date: 20220321