WO2017190664A1 - Utilisation d'arncr et d'arncr modifié obtenus par chimiosynthèse dans des systèmes d'édition de gènes crispr/cpf1 - Google Patents

Utilisation d'arncr et d'arncr modifié obtenus par chimiosynthèse dans des systèmes d'édition de gènes crispr/cpf1 Download PDF

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WO2017190664A1
WO2017190664A1 PCT/CN2017/082968 CN2017082968W WO2017190664A1 WO 2017190664 A1 WO2017190664 A1 WO 2017190664A1 CN 2017082968 W CN2017082968 W CN 2017082968W WO 2017190664 A1 WO2017190664 A1 WO 2017190664A1
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crrna
cell group
plasmid
modified
gene
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PCT/CN2017/082968
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Chinese (zh)
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王德华
张佩琢
徐明亮
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苏州吉玛基因股份有限公司
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Priority claimed from CN201610292302.XA external-priority patent/CN105907785B/zh
Priority claimed from CN201610703716.7A external-priority patent/CN106244591A/zh
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    • 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
    • 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

Definitions

  • the invention relates to the field of biotechnology, and particularly relates to the application of chemically synthesized crRNA and modified crRNA in the CRISPR/Cpf1 gene editing system.
  • Gene editing is a technique for precise modification at the genomic level, which can perform gene-based deletion (InDel) mutations, gene-site insertion mutations, simultaneous multi-site mutations, and censoring of small fragments.
  • Gene editing technology can be used for gene function and disease pathogenesis research, construction of disease animal models, biological therapy, genetic and tumor-related disease research, integration of viral disease research and improvement of agricultural and livestock species.
  • Gene editing technology is a tool that fundamentally changes the DNA of species genetic material, and has extremely wide application value and development prospects.
  • Zinc finger nuclease ZFN
  • transcriptional activator-like effector nuclease TALEN
  • CRISPR/Cas9 systems are three major gene editing technologies, essentially using non-homologous end-linking pathway (NHEJ) repair and homologous recombination. (HR) repair, targeted recognition of specific DNA and alteration of DNA sequence by endonuclease.
  • NHEJ repair causes gene insertion or deletion mutations, resulting in gene frameshift mutations to achieve coding protein gene knockout. If two double-strand breaks in the vicinity of the genome, NHEJ repair can cause genomic fragment deletion.
  • ZFN zinc finger nuclease
  • TALEN transcriptional activator-like effector nuclease
  • CRISPR/Cas9 CRISPR/Cas9 system
  • the FokI structure needs to form a dimer to play the DSB cleavage function, so both ZFNs and TALEN need to express two DNA targeting-FokI endonuclease structural fusion proteins.
  • the CRISPR/Cas9 system is an acquired immune system found in most bacteria (about 40%) and archaea (about 90%), which can be used to destroy or fight against foreign plastids or phage, and to remain in their own genome.
  • the next foreign gene fragment acts as a "memory".
  • the expressed Cas9 nuclease cleaves the foreign gene containing the PAM and is resistant to foreign resources under the guidance of the crRNA and tracrRNA containing the memory fragment.
  • the immune function of DNA fragment invasion Editing the biological genome with the CRISPR/Cas9 system can cause different forms of deletion or insertion at the target fragment, and has been successfully applied to human cell lines, zebrafish, rats, mice, fruit flies and other organisms.
  • the recognition of DNA sequences by proteins in the CRISPR/Cas9 system is much more precise, reducing the probability of off-target cleavage and reducing cytotoxicity, and the construction of the CRISPR/Cas9 system only needs to be designed to complement the target sequence.
  • the gRNA can be simpler and cheaper, which greatly improves the efficiency and simplicity of gene manipulation.
  • the CRISPR/Cas9 system also has some shortcomings.
  • the Cas9 protein cannot cleave any sequence, and its target 3' end must contain a PAM sequence (eg, the SpCas9 protein requires the PAM sequence to be NGG).
  • CRISPR/Cpf1 The newly discovered CRISPR/Cpf1 system (Zetsche B, Gootenberg JS et al. Cpf1is a single RNA-guided endonuclease of a class 2CRISPR-Cas system.Cell.2015Oct 22;163(3):759-71.) and CRISPR/Cas9
  • the system belongs to the CRISPR-Cas Class2 system, but the former only needs one.
  • Gene editing with shorter crRNAs has the potential to enable simpler, more accurate genome engineering operations.
  • the technical problem to be solved by the present invention is how to perform gene editing.
  • the present invention firstly provides the application of the CRISPR/Cpf1 system in gene editing; the CRISPR/Cpf1 system includes q1) or q2) or q3): q1) chemically synthesized crRNA; q2) chemical synthesis And a modified crRNA; q3) a vector expressing a crRNA.
  • the vector may be a recombinant vector obtained by inserting the coding DNA of the crRNA into a multiple cloning site of a backbone vector.
  • the backbone vector can be a cloning vector.
  • the cloning vector may specifically be a plasmid pU6gRNA produced by Suzhou Gemma Gene Co., Ltd.
  • the vector may specifically be a fragment between the restriction endonuclease BbsI of the plasmid pU6gRNA and the EcoRI recognition sequence (the plasmid pU6gRNA is cleaved into a large fragment by the restriction enzymes BbsI and EcoRI) And a small fragment, the DNA is the small fragment) replaced with the recombinant vector obtained by encoding the DNA of the crRNA.
  • the CRISPR/Cpf1 system may be c1) or c2) or c3) or c4): c1) LbCRISPR/Cpf1 system; c2) Lb2 CRISPR/Cpf1 system; c3) FnCRISPR/Cpf1 system; c4) AsCRISPR/Cpf1 system.
  • the AsCRISPR/Cpf1 system is derived from Acidaminococcus sp. BV3L6, which expresses the AsCpfl protein.
  • the FnCRISPR/Cpf1 system is from Francisella_novicida, which expresses the FnCpfl protein.
  • the LbCRISPR/Cpf1 system is from Lachnospiraceae bacterium ND2006, which expresses the LbCpf1 protein.
  • the Lb2 CRISPR/Cpf1 system is derived from Lachnospiraceae_bacterium_MA2020, which expresses the Lb2Cpf1 protein.
  • the AsCpf1 protein may be h1) or h2) or h3): h1) the amino acid sequence is the protein shown in SEQ ID NO: 6 in the sequence listing; h2) the fusion protein obtained by ligating the N-terminus or/and C-terminus of h1); H3) A protein having the same function obtained by subjecting the amino acid sequence shown by the sequence 6 in the sequence listing to substitution and/or deletion and/or addition of one or several amino acid residues.
  • the FnCpf1 protein may be i1) or i2) or i3): i1) the amino acid sequence is the protein shown in SEQ ID NO: 5 in the sequence listing; i2) the fusion protein obtained by ligating the N-terminus or/and C-terminus of i1); I3) A protein having the same function obtained by subjecting the amino acid sequence shown by the sequence 5 in the sequence listing to substitution and/or deletion and/or addition of one or several amino acid residues.
  • the LbCpf1 protein may be j1) or j2) or j3): j1) the amino acid sequence is the protein shown in SEQ ID NO: 3 in the sequence listing; j2) the fusion protein obtained by ligating the N-terminus or/and the C-terminus of j1); J3) A protein having the same function obtained by subjecting the amino acid sequence shown by the sequence 3 in the sequence listing to substitution and/or deletion and/or addition of one or several amino acid residues.
  • the Lb2Cpf1 protein may be k1) or k2) or k3): k1) the amino acid sequence is the protein shown in SEQ ID NO: 4 in the sequence listing; k2) the fusion protein obtained by ligating the N-terminus or/and C-terminus of k1); K3) A protein having the same function obtained by subjecting the amino acid sequence shown in SEQ ID NO: 4 to substitution and/or deletion and/or addition of one or several amino acid residues.
  • the present invention also provides a method of directed editing of a genome.
  • the method for directly editing a genome may be the method one, and may include the following steps:
  • the target gene may specifically be the hAAVS1 gene (Gene ID: 54776) or the THUMPD3-AS1 gene (Gene ID: 440944).
  • the target sequence I is selected based on the nucleotide sequence of the hAAVS1 gene, and the nucleotide sequence of the target sequence I may specifically be: 5'-TCTGTCCCCTCCACCCCACAGTGG-3'.
  • the target sequence II is selected based on the nucleotide sequence of the THUMPD3-AS1 gene, and the nucleotide sequence of the target sequence II may specifically be: 5'-GAGAACAAGCGCCTCCCACCCACA-3'.
  • the Cpf1 protein may be the AsCpf1 protein or the FnCpf1 protein.
  • the crRNA is chemically synthesized and modified to obtain a modified crRNA.
  • the modified crRNA may specifically be any one of e1)-e16):
  • the gene encoding the AsCpfl protein can be introduced into the receptor in the form of a plasmid.
  • the plasmid may specifically be the plasmid pcDNA3.1-hAsCpf1 of Addgene.
  • the receptor can be a 293T cell.
  • the gene encoding the FnCpfl protein can be introduced into the receptor in the form of a plasmid.
  • the plasmid may specifically be Addgene's plasmid pcDNA3.1-hFnCpf1.
  • the receptor can be a 293T cell.
  • the method for direct editing of the genome provided by the present invention may be the second method, and may include the following steps:
  • the target gene may specifically be the hAAVS1 gene and the THUMPD3-AS1 gene.
  • the target sequence I is selected based on the nucleotide sequence of the hAAVS1 gene.
  • the target sequence II is selected based on the nucleotide sequence of the THUMPD3-AS1 gene.
  • the Cpf1 protein may be the AsCpf1 protein, the FnCpf1 protein, the LbCpf1 protein or the Lb2Cpf1 protein.
  • the chemically synthesized crRNA may specifically be any one of x9)-x14):
  • a gene encoding said LbCpfl protein is introduced into said receptor.
  • the gene encoding the LbCpfl protein can be introduced into the receptor in the form of a plasmid.
  • the plasmid may specifically be the plasmid pcDNA3.1-hLbCpf1 of Addgene.
  • the receptor can be a 293T cell.
  • Said x11) or said x12) or said x13) or said x14 in particular, a gene encoding said Lb2Cpf1 protein introduced into said receptor.
  • the gene encoding the Lb2Cpf1 protein can be introduced into the receptor in the form of a plasmid.
  • the plasmid may specifically be the plasmid pcDNA3.1-hLb2Cpf1 of Addgene.
  • the receptor can be a 293T cell.
  • the method for direct editing of the genome provided by the present invention may be the third method, which may include the following steps:
  • the recombinant vector may be a recombinant vector obtained by inserting the coding DNA of the crRNA into a multiple cloning site of a skeleton vector.
  • the backbone vector can be a cloning vector.
  • the cloning vector may specifically be a plasmid pU6gRNA produced by Suzhou Gemma Gene Co., Ltd.
  • the recombinant vector may specifically be a fragment between the restriction endonuclease BbsI and the EcoRI recognition sequence of the plasmid pU6gRNA (the plasmid pU6gRNA is cleaved into a large fragment and a restriction enzyme BbsI and EcoRI) A small fragment, the DNA is a small fragment) replaced with a recombinant vector obtained by encoding the DNA of the crRNA.
  • the target gene may specifically be the hAAVS1 gene and the THUMPD3-AS1 gene.
  • the target sequence I is selected based on the nucleotide sequence of the hAAVS1 gene.
  • the target sequence II is selected based on the nucleotide sequence of the THUMPD3-AS1 gene.
  • the Cpf1 protein It may be the AsCpfl protein, the FnCpfl protein, the LbCpfl protein or the Lb2Cpfl protein.
  • the “designing the crRNA according to the target gene expected to be oriented and edited in the receptor genome” may specifically be any one of x1)-x8):
  • the recombinant vector expressing the x1) or the x2) is specifically introduced into the receptor with a gene encoding the AsCpf1 protein.
  • the gene encoding the AsCpfl protein can be introduced into the receptor in the form of a plasmid.
  • the plasmid may specifically be the plasmid pcDNA3.1-hAsCpf1 of Addgene.
  • the receptor can be a 293T cell.
  • a recombinant vector expressing the x3) or the x4), specifically, a gene encoding the FnCpf1 protein is introduced into the receptor.
  • the gene encoding the FnCpfl protein can be introduced into the receptor in the form of a plasmid.
  • the plasmid may specifically be the plasmid pcDNA3.1-hFnCpf1 of Addgene.
  • the receptor can be a 293T cell.
  • a recombinant vector expressing the x5) or the x6), specifically, a gene encoding the LbCpf1 protein is introduced into the receptor.
  • the gene encoding the LbCpfl protein can be introduced into the receptor in the form of a plasmid.
  • the plasmid may specifically be the plasmid pcDNA3.1-hLbCpf1 of Addgene.
  • the receptor can be a 293T cell.
  • a recombinant vector expressing the x7) or the x8), specifically, a gene encoding the Lb2Cpf1 protein is introduced into the receptor.
  • the gene encoding the Lb2Cpf1 protein can be introduced into the receptor in the form of a plasmid.
  • the plasmid may specifically be the plasmid pcDNA3.1-hLb2Cpf1 of Addgene.
  • the receptor can be a 293T cell.
  • the present invention also provides a CRISPR/Cpf1 system for directed editing of a genome.
  • the invention provides a CRISPR/Cpf1 system for directional editing of genomes, which comprises q1) or q2) or q3): q1) chemically synthesized crRNA; q2) chemically synthesized and modified crRNA; q3) vector expressing crRNA .
  • the vector in the above system, in the q3), may be a recombinant vector obtained by inserting the coding DNA of the crRNA into a multiple cloning site of a backbone vector.
  • the backbone vector can be a cloning vector.
  • the cloning vector may specifically be a plasmid pU6gRNA produced by Suzhou Gemma Gene Co., Ltd.
  • the vector in the above system, in the q3), may specifically be a fragment between the restriction endonuclease BbsI of the plasmid pU6gRNA and the EcoRI recognition sequence (the plasmid pU6gRNA is cleaved into a large fragment by the restriction enzymes BbsI and EcoRI) And a small fragment, the DNA is the small fragment) replaced with the recombinant vector obtained by encoding the DNA of the crRNA.
  • the CRISPR/Cpf1 system may be any of the LbCRISPR/Cpf1 systems described above or any of the Lb2 CRISPR/Cpf1 systems described above or any of the FnCRISPR/Cpf1 systems described above or any of the above One of the AsCRISPR/Cpf1 systems.
  • any of the above modifications may be by adding a deoxyribonucleic acid and/or a ribonucleotide at the 5' end and/or the 3' end of the crRNA; the deoxyribonucleic acid is modified or unmodified; The ribonucleic acid is modified or unmodified.
  • any of the above modifications may be one of p1)-p7): p1) deoxyribonucleic acid modification; p2) methoxy modification of ribonucleotides; p3) thio modification of ribonucleotides; p4 a methoxythio modification of a ribonucleotide; p5) a F modification of a ribonucleotide; p6) a locked nucleotide modification of a ribonucleotide; and p7) a thio modification of a deoxyribonucleic acid.
  • the method for deoxyribonucleic acid modification according to any of the above methods is to add 1 to 3 deoxyribonucleic acids to the 5' end of the crRNA and/or to add 1 to 3 deoxyribonucleic acids to the 3' end.
  • the methoxy modification of any of the above ribonucleotides is carried out by adding 1 to 3 methoxy-modified ribonucleotides at the 5' end of the crRNA and/or adding 1 to 3 at the 3' end. Oxygen-modified ribonucleotides.
  • a method for thio modification of any of the above ribonucleotides is to add 1 to 3 thio-modified ribonucleotides at the 5' end of the crRNA and/or to add 1 to 3 sulphur at the 3' end Substituted modified ribonucleotides.
  • the methoxythio modification of any of the above ribonucleotides is carried out by adding 1 to 3 methoxythio-modified ribonucleotides to the 5' end of the crRNA and/or increasing the 3' end by 1 ⁇ 3 methoxythio modified ribonucleotides.
  • the F modification of any of the above ribonucleotides is carried out by adding 1 to 3 F-modified ribonucleotides at the 5' end of the crRNA and/or adding 1 to 3 F-modified at the 3' end. Ribonucleotides.
  • the method for modifying a locked nucleotide of any of the above ribonucleotides is to add 1 to 3 locked nucleotide-modified ribonucleotides at the 5' end of the crRNA and/or to increase the 3' end by 1 ⁇ 3 locked nucleotide modified ribonucleotides.
  • the thio modification of any of the above-mentioned deoxyribonucleic acids is carried out by adding 1 to 3 thio-modified deoxyribonucleic acids at the 5' end of the crRNA and/or adding 1 to 3 thio modifications at the 3' end. DNA.
  • the method for deoxyribonucleic acid modification of any of the above may specifically be any one of a1)-a12):
  • deoxyribonucleic acids may specifically be thymidine deoxynucleotides.
  • Any of the above ribonucleotides is a uridine ribonucleotide.
  • modified crRNAs Any of the "modified crRNAs" described above may specifically be a chemically synthesized and modified crRNA.
  • the chemically synthesized crRNA of any of the above is not introduced by a vector expressing a crRNA.
  • the crRNA expressed by the recombinant vector has certain gene editing ability in the AsCRISPR/Cpf1 system, FnCRISPR/Cpf1 system, LbCRISPR/Cpf1 system and Lb2 CRISPR/Cpf1 system, and the gene editing ability of each of the above CRISPR/Cpf1 systems is :AsCRISPR/Cpf1 system>FnCRISPR/Cpf1 system>LbCRISPR/Cpf1 system>Lb2CRISPR/Cpf1 system; chemically synthesized crRNA and chemically synthesized and modified crRNA for AsCRISPR/Cpf1 system or FnCRISPR/Cpf1 system cause hAAVS1 gene and THUMPD3 -
  • the mutation of the AS1 gene has certain gene editing ability.
  • the chemically synthesized crRNA can be directly transfected, is easier to manipulate, more controllable, and facilitates chemical modification than transfection by constructing a recombinant vector.
  • the chemically synthesized and modified crRNA has a stronger gene editing ability than the chemically synthesized crRNA. Therefore, the recombinant vector-expressed crRNA and/or chemically synthesized crRNA and/or chemically synthesized and modified crRNA for the CRISPR/Cpf1 system have important application value in gene editing.
  • Figure 1 shows the results of T7E1 mutation detection in step 3 of Example 1.
  • Figure 3 is a graph showing the results of T7E1 mutation detection in step 2 of Example 2.
  • Figure 4 is the sequencing result of 4 in the second step of Example 2.
  • Figure 5 is a graph showing the results of T7E1 mutation detection in step 3 of Example 3.
  • Figure 6 is the sequencing result of 4 in the third step of Example 3.
  • Plasmid pcDNA3.1-hAsCpf1, plasmid pcDNA3.1-hFnCpf1, plasmid pcDNA3.1-hLbCpf1 and plasmid pcDNA3.1-hLb2Cpf1 are products of Addgene, hereinafter, plasmid pcDNA3.1-hAsCpf1 is abbreviated as plasmid Y1681, plasmid pcDNA3 .1-hFnCpf1 is abbreviated as plasmid Y1682, plasmid pcDNA3.1-hLbCpf1 is abbreviated as plasmid Y1683, and plasmid pcDNA3.1-hLb2Cpf1 is abbreviated as plasmid Y1684.
  • Plasmid pU6gRNA is a product of Suzhou Gemma Gene Co., Ltd., plasmid pU6gRNA (loop) nucleus
  • the nucleotide sequence is shown in Sequence Listing 1.
  • the plasmid pU6gRNA is abbreviated as Y523.
  • the 293T cell is a cell bank product of the Chinese Academy of Sciences, catalog number GNHu17.
  • DMEM medium and FBS are products of Gibco.
  • the cell plate is a product of Corning. Max enzyme is Vazyme's product, the product number is P505.
  • the Genomic DNA Extraction kit is a product of Takara, catalog number #9765.
  • Trypsin-EDTA Solution is Hyclone, Inc., item number SH30042.02.
  • the PBS buffer was obtained by diluting PBS (10 ⁇ ) to 10 volumes in ultrapure water; PBS (10 ⁇ ) was produced by Biotech (Shanghai) Co., Ltd., and the product number was E607016.
  • the OPTI-MEM medium is a product of Gibco, catalog number #31985-070.
  • Lipofectamine 2000 is a product of thermo fisher-invitrogen, article number 11668-027.
  • T7E1 is a component of the T7E1 mutation detection kit; the T7E1 mutation detection kit is a product of Suzhou Jima Gene Co., Ltd.
  • DNA Marker is a product of Thermo Fisher Company under the product name GeneRuler DNA Ladder Mix, item number SM0331.
  • 10 x annealing buffer pH 8.0 containing 10 mM EDTA ⁇ 2Na, 1000 mM NaCl, 100 mM Tris-HCl buffer.
  • Example 1 Detection of gene editing ability of AsCRISPR/Cpf1 system, FnCRISPR/Cpf1 system, LbCRISPR/Cpf1 system and Lb2 CRISPR/Cpf1 system
  • the hAAVS1 gene (Gene ID: 54776) and the THUMPD3-AS1 gene (Gene ID: 440944) were selected as target genes for detecting the gene editing ability of the AsCRISPR/Cpf1 system, the FnCRISPR/Cpf1 system, the LbCRISPR/Cpf1 system, and the Lb2 CRISPR/Cpf1 system.
  • the AsCRISPR/Cpf1 system is derived from Acidaminococcus_sp.BV3L6, which expresses the AsCpf1 protein shown in SEQ ID NO: 6 in the sequence listing;
  • the FnCRISPR/Cpf1 system is from Francisella_novicida, which expresses the FnCpf1 protein shown in SEQ ID NO: 5 in the sequence listing;
  • the LbCRISPR/Cpf1 system is derived from Lachnospiraceae bacterium.
  • the Lb2 CRISPR/Cpf1 system is derived from Lachnospiraceae_bacterium_MA2020, which expresses the Lb2Cpf1 protein shown in SEQ ID NO: 4 in the Sequence Listing.
  • target sequence I is selected based on the nucleotide sequence of the hAAVS1 gene
  • target sequence II is selected based on the nucleotide sequence of the THUMPD3-AS1 gene.
  • sequences of target sequence I and target sequence II are as follows:
  • Target sequence I 5'-TCTGTCCCCTCCACCCCACAGTGG-3';
  • Target sequence II 5'-GAGAACAAGCGCCTCCCACCCACA-3'.
  • Plasmid pU6gRNA was digested with restriction endonucleases BbsI and EcoRI, and a vector backbone of about 2955 bp was recovered.
  • primer Y1640-S by Suzhou Jima Gene Co., Ltd.: (Underlined as the As crRNA backbone sequence, double underlined for target sequence I, wavy line is the termination sequence) and primer Y1640-A: (Underlined as the As crRNA backbone sequence, double underlined for the target sequence I, the wavy line is the termination sequence), the primer Y1640-S and the primer Y1640-A were diluted to 100 ⁇ M with deionized water to obtain the primer Y1640-S dilution and Primer Y1640-A dilution; then an annealing reaction to form DNA molecule I.
  • Annealing system 5 ⁇ L of primer Y1640-S dilution, 5 ⁇ L of primer Y1640-S dilution, 35 ⁇ L of deionized water, and 5 ⁇ L of 10 ⁇ annealing buffer (inclusive).
  • Annealing procedure 99 ° C 10 min, 85 ° C 5 min, 80 ° C 5 min, 75 ° C 5 min, 70 ° C 5 min, 16 ° C preservation.
  • the plasmid Y1640 was structurally described as follows: a fragment between the restriction endonuclease BbsI and EcoRI recognition sequences of plasmid pU6gRNA (plasmid pU6gRNA was cut into a large fragment and a small fragment by restriction endonucleases BbsI and EcoRI) , the DNA is the small fragment) replaced with DNA molecule I.
  • the nucleotide sequence of plasmid Y1640 is shown in Sequence Listing 2 of the Sequence Listing.
  • the preparation method of DNA molecule II is as follows: Synthetic primer Y1641-S was synthesized by Suzhou Jima Gene Co., Ltd.: (Underlined as the As crRNA backbone sequence, double underlined for target sequence II, wavy line is the termination sequence) and primer Y1641-A: (Underlined as the As crRNA backbone sequence, double underlined for target sequence II, wavy line is the termination sequence), and the primer Y1641-S and the primer Y1641-A were diluted to 100 ⁇ M with deionized water to obtain the primer Y1641-S dilution and Primer Y1641-A dilution; then an annealing reaction to form DNA molecule II.
  • Annealing system 5 ⁇ L of primer Y1641-S dilution solution, 5 ⁇ L of primer Y1641-S dilution solution, 35 ⁇ L of deionized water, and 10 ⁇ L of annealing buffer (inclusive).
  • Annealing procedure 99 ° C 10 min, 85 ° C 5 min, 80 ° C 5 min, 75 ° C 5 min, 70 ° C 5 min, 16 ° C preservation.
  • plasmid Y1641 was described as follows: a fragment between the restriction endonuclease BbsI and EcoRI recognition sequences of plasmid pU6gRNA (plasmid pU6gRNA was cut into a large fragment and a small fragment by restriction endonucleases BbsI and EcoRI) , the DNA is the small fragment) replaced with DNA molecule II.
  • the preparation method of DNA molecule III is as follows: Synthetic primer Y1642-S was synthesized by Suzhou Jima Gene Co., Ltd.: (Underlined as Fn crRNA backbone sequence, double underlined for target sequence I, wavy line is the termination sequence) and primer Y1642-A: (Underlined as the Fn crRNA backbone sequence, double underlined for the target sequence I, the wavy line is the termination sequence), the primer Y1642-S and the primer Y1642-A were diluted to 100 ⁇ M with deionized water to obtain the primer Y1642-S dilution and Primer Y1642-A dilution; then an annealing reaction to form DNA molecule III.
  • Annealing system 5 ⁇ L of primer Y1642-S dilution, 5 ⁇ L of primer Y1642-S dilution, 35 ⁇ L of deionized water, and 10 ⁇ L of annealing buffer (inclusive).
  • Annealing procedure 99 ° C 10 min, 85 ° C 5 min, 80 ° C 5 min, 75 ° C 5 min, 70 ° C 5 min, 16 ° C preservation.
  • the plasmid Y1642 was structurally described as follows: a fragment between the restriction endonuclease BbsI and EcoRI recognition sequences of plasmid pU6gRNA (plasmid pU6gRNA was cut into a large fragment and a small fragment by restriction endonucleases BbsI and EcoRI) , the DNA is the small fragment) replaced with DNA molecule III.
  • the preparation method of DNA molecule IV is as follows: Synthetic primer Y1643-S was synthesized by Suzhou Jima Gene Co., Ltd.: (Underlined as Fn crRNA backbone sequence, double underlined for target sequence II, wavy line is the termination sequence) and primer Y1643-A: (Underlined as Fn crRNA backbone sequence, double underlined for target sequence II, wavy line is termination sequence), dilute primer Y1643-S and primer Y1643-A to 100 ⁇ M with deionized water, respectively, to obtain primer Y1643-S dilution and Primer Y1643-A dilution; then an annealing reaction to form DNA molecule III.
  • Annealing system 5 ⁇ L of primer Y1643-S dilution, 5 ⁇ L of primer Y1643-S dilution, 35 ⁇ L of deionized water, and 5 ⁇ L of 10 ⁇ annealing buffer (inclusive).
  • Annealing procedure 99 ° C 10 min, 85 ° C 5 min, 80 ° C 5 min, 75 ° C 5 min, 70 ° C 5 min, 16 ° C preservation.
  • plasmid Y1643 a fragment between the restriction endonuclease BbsI and EcoRI recognition sequences of plasmid pU6gRNA (plasmid pU6gRNA was cut into a large fragment and a small fragment by restriction endonucleases BbsI and EcoRI) , the DNA is the small fragment) replaced with DNA molecule IV.
  • the preparation method of DNA molecule V is as follows: Synthetic primer Y1644-S was synthesized by Suzhou Jima Gene Co., Ltd.: (Underlined as Lb crRNA backbone sequence, double underlined for target sequence I, wavy line is the termination sequence) and primer Y1644-A:
  • Annealing system primer ⁇ 1644-S dilution 5 ⁇ L, primer Y1644-S dilution 5 ⁇ L, deionized water 35 ⁇ L, 10 ⁇ annealing buffer (inclusive) 5 ⁇ L.
  • Annealing procedure 99 ° C 10 min, 85 ° C 5 min, 80 ° C 5 min, 75 ° C 5 min, 70 ° C 5 min, 16 ° C preservation.
  • the plasmid Y1644 was structurally described as follows: a fragment between the restriction endonuclease BbsI and EcoRI recognition sequences of plasmid pU6gRNA (plasmid pU6gRNA was cut into a large fragment and a small fragment by restriction endonucleases BbsI and EcoRI) , the DNA is the small fragment) replaced with the DNA molecule V.
  • the preparation method of DNA molecule VI is as follows: Synthetic primer Y1645-S was synthesized by Suzhou Jima Gene Co., Ltd.: (Underlined as Lb crRNA backbone sequence, double underlined for target sequence II, wavy line is the termination sequence) and primer Y1645-A:
  • Annealing system primer ⁇ 1645-S dilution 5 ⁇ L, primer Y1645-S dilution 5 ⁇ L, deionized water 35 ⁇ L, 10 ⁇ annealing buffer (inclusive) 5 ⁇ L.
  • Annealing procedure 99 ° C 10 min, 85 ° C 5 min, 80 ° C 5 min, 75 ° C 5 min, 70 ° C 5 min, 16 ° C preservation.
  • the plasmid Y1645 was structurally described as follows: a fragment between the restriction endonuclease BbsI and EcoRI recognition sequences of plasmid pU6gRNA (plasmid pU6gRNA was cut into a large fragment and a small fragment by restriction endonucleases BbsI and EcoRI) , the DNA is the small fragment) replaced with the DNA molecule VI.
  • the preparation method of DNA molecule VII is as follows: Synthetic primer Y1646-S was synthesized by Suzhou Jima Gene Co., Ltd.: (Underlined as Lb2crRNA backbone sequence, double underlined for target sequence I, wavy line is the termination sequence) and primer Y1646-A: (The single underlined is the Lb2crRNA backbone sequence, double underlined for the target sequence I, and the wavy line is the termination sequence).
  • the primer Y1646-S and the primer Y1646-A were diluted to 100 ⁇ M with deionized water to obtain the primer Y1646-S dilution and primer. Y1646-A dilution; then an annealing reaction to form DNA molecule VII.
  • Annealing system primer ⁇ 1646-S dilution 5 ⁇ L, primer Y1646-S dilution 5 ⁇ L, deionized water 35 ⁇ L, 10 ⁇ annealing buffer (inclusive) 5 ⁇ L.
  • Annealing procedure 99 ° C 10 min, 85 ° C 5 min, 80 ° C 5 min, 75 ° C 5 min, 70 ° C 5 min, 16 ° C preservation.
  • plasmid Y1646 was structurally described as follows: a fragment between the restriction endonuclease BbsI and EcoRI recognition sequences of plasmid pU6gRNA (plasmid pU6gRNA was cleaved into a large fragment and a small fragment by restriction endonucleases BbsI and EcoRI) , the DNA is the small fragment) replaced with DNA molecule VII.
  • the preparation method of DNA molecule VIII is as follows: Synthetic primer Y1647-S was synthesized by Suzhou Jima Gene Co., Ltd.: (Single underlined is the Lb2crRNA backbone sequence, double underlined for target sequence II, wavy line is the termination sequence) and primer Y1647-A: (Underlined as Lb2crRNA backbone sequence, double underlined for target sequence II, wavy line is termination sequence), primer Y1647-S and primer Y1647-A were diluted to 100 ⁇ M with deionized water to obtain primer Y1647-S dilution and primer. Y1647-A dilution; then an annealing reaction to form DNA molecule VIII.
  • Annealing system 5 ⁇ L of primer Y1647-S dilution, 5 ⁇ L of primer Y1647-S dilution, 35 ⁇ L of deionized water, and 5 ⁇ L of 10 ⁇ annealing buffer (inclusive).
  • Annealing procedure 99 ° C 10 min, 85 ° C 5 min, 80 ° C 5 min, 75 ° C 5 min, 70 ° C 5 min, 16 ° C preservation.
  • the plasmid Y1647 was structurally described as follows: a fragment between the restriction endonuclease BbsI and EcoRI recognition sequences of plasmid pU6gRNA (plasmid pU6gRNA was cut into a large fragment and a small fragment by restriction endonucleases BbsI and EcoRI) , the DNA is the small fragment) replaced with DNA molecule VIII.
  • step 2 After completing step 1, add 1 mL of Trypsin-EDTA Solution to the culture dish, mix, then aspirate the liquid phase, and let stand at 37 ° C for 1-2 min.
  • step 2 2 mL of DMEM medium containing 10% by volume of FBS was added to the culture dish, and blown to form a single cell suspension.
  • step 3 the single cell suspension was inoculated into a 6-well plate, and about 2 ⁇ 10 5 293T cells were inoculated into each well, and cultured in a 37° C., 5% CO 2 incubator for 24 hours.
  • the medium in the 6-well plate was changed to OPTI-MEM medium, and then 4 ⁇ g of plasmid Y1681 and 4 ⁇ g of plasmid Y1640 were added for co-transfection (in the process of co-transfection, the transfection reagent was lipofectamine). 2000, the medium is OPTI-MEM medium, the process of co-transfection is referred to Lipofectamin2000 manual), then incubated in 37 ° C, 5% CO 2 incubator for 6 h, then replaced with new OPTI-MEM medium, 37 ° C, 5 Incubation was continued for 18 h in a %CO 2 incubator.
  • step 7 After 48 hours from the completion of step 6, the cells were collected and then washed once with 1 mL of PBS.
  • step 7 0.5 mL of Trypsin-EDTA Solution was added to the culture dish, mixed, and then the liquid phase was aspirated, and allowed to stand at 37 ° C for 1 to 2 minutes.
  • step 8 After completion of step 8, 1 mL of DMEM medium containing 10% by volume of FBS was added to the culture dish, and a single cell suspension was formed by pipetting; the single cell suspension was centrifuged at 1000 rpm for 3 minutes to obtain a precipitate 1.
  • step 9 After completion of step 9, 1 mL of PBS was added to the precipitate, and the mixture was centrifuged at 1000 rpm for 3 minutes to obtain a precipitate 2.
  • Precipitate 2 is the 293T cell group co-transfected with plasmid Y1681 and plasmid Y1640, referred to as Y1681-Y1640-293T cell group.
  • the medium in the 6-well plate was changed to OPTI-MEM medium, and then 4 ⁇ g of plasmid Y1681 was added for transfection (in the process of co-transfection, the transfection reagent was lipofectamine 2000, and the medium was OPTI-MEM medium, co-transfection step reference Lipofectamin2000 instructions), then incubated in 37 ° C, 5% CO 2 incubator for 6h, then replaced with new OPTI-MEM medium, 37 ° C, 5% CO 2 incubator Continue to culture for 18h.
  • the precipitate obtained in step 10 is the 293T cell group transfected with plasmid Y1681, referred to as Y1681-293T cell group.
  • the plasmid Y1640 was replaced with the plasmid Y1641, and the other steps were unchanged, and the Y1681-Y1641-293T cell group was obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1642, and the other steps were unchanged, and the Y1681-Y1642-293T cell group was obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1643, and the other steps were unchanged, and the Y1681-Y1643-293T cell group was obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1644, and the other steps were unchanged, and the Y1681-Y1644-293T cell group was obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1645, and the other steps were unchanged, and the Y1681-Y1645-293T cell group was obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1646, and the other steps were unchanged, and the Y1681-Y1646-293T cell group was obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1647, and the other steps were unchanged, and the Y1681-Y1647-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, the plasmid Y1683 and the plasmid Y1684, respectively, and the other steps were unchanged, and the Y1682-Y1640-293T cell group, the Y1683-Y1640-293T cell group and the Y1684-Y1640 were obtained. -293T cell group.
  • the plasmid Y1681 was replaced with the plasmid Y1682, the plasmid Y1683 and the plasmid Y1684, respectively, and the other steps were unchanged, and the Y1682-293T cell group, the Y1683-293T cell group and the Y1684-293T cell group were obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1641, and the plasmid Y1681 was replaced with the plasmid Y1682, the plasmid Y1683 and the plasmid Y1684, respectively, and the other steps were unchanged, and the Y1682-Y1641-293T cell group and Y1683-Y1641 were obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1642, and the plasmid Y1681 was replaced with the plasmid Y1682, the plasmid Y1683 and the plasmid Y1684, respectively, and the other steps were unchanged, and the Y1682-Y1642-293T cell group and the Y1683-Y1642 were obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1643, and the plasmid Y1681 was replaced with the plasmid Y1682, the plasmid Y1683 and the plasmid Y1684, respectively, and the other steps were unchanged, and the Y1682-Y1643-293T cell group and the Y1683-Y1643 were obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1644, and the plasmid Y1681 was replaced with the plasmid Y1682, the plasmid Y1683 and the plasmid Y1684, respectively, and the other steps were unchanged, and the Y1682-Y1644-293T cell group and the Y1683-Y1644 were obtained. -293T cell group and Y1684-Y1644-293T cell group.
  • the plasmid Y1640 was replaced with the plasmid Y1645, and the plasmid Y1681 was replaced with the plasmid Y1682, the plasmid Y1683 and the plasmid Y1684, respectively, and the other steps were unchanged, and the Y1682-Y1645-293T cell group and the Y1683-Y1645 were obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1646, and the plasmid Y1681 was replaced with the plasmid Y1682, the plasmid Y1683 and the plasmid Y1684, respectively, and the other steps were unchanged, and the Y1682-Y1646-293T cell group and the Y1683-Y1646 were obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1647, and the plasmid Y1681 was replaced with the plasmid Y1682, the plasmid Y1683 and the plasmid Y1684, respectively, and the other steps were unchanged, and the Y1682-Y1647-293T cell group and the Y1683-Y1647 were obtained. -293T cell group and Y1684-Y1647-293T cell group.
  • the genomic DNA of each cell group obtained in the step 1 was separately extracted with a Genomic DNA Extraction kit.
  • step (1) Y1681-Y1640-293T cell group, Y1682-Y1640-293T cell group, Y1683-Y1640-293T cell group and Y1684-Y1640-293T cell group extracted by step (1), Y1681 -Y1642-293T cell group, Y1682-Y1642-293T cell group, Y1683-Y1642-293T cell group, Y1684-Y1642-293T cell group, Y1681-Y1644-293T cell group, Y1682-Y1644-293T cell group, Y1683-Y1644
  • the genomic DNA of the -293T cell group, Y1684-Y1644-293T cell group, Y1681-Y1646-293T cell group, Y1682-Y1646-293T cell group, Y1683-Y1646-293T cell group or Y1684-Y1646-293T cell group is used as a template.
  • a primer consisting of hAAV-F: 5'-GGGTCACCTCTCACTCCTTTCAT-3' and hAAV-R: 5'-ATCCTCTCTGGCTCCATCGTAAG-3' was subjected to PCR amplification using Max enzyme to obtain a 475 bp PCR amplification product A.
  • step (1) Y1681-Y1641-293T cell group, Y1682-Y1641-293T cell group, Y1683-Y1641-293T cell group and Y1684-Y1641-293T cell group extracted by step (1), Y1681 -Y1643-293T cell group, Y1682-Y1643-293T cell group, Y1683-Y1643-293T cell group, Y1684-Y1643-293T cell group, Y1681-Y1645-293T cell group, Y1682-Y1645-293T cell group, Y1683-Y1645
  • the genomic DNA of the -293T cell group, Y1684-Y1645-293T cell group, Y1681-Y1647-293T cell group, Y1682-Y1647-293T cell group, Y1683-Y1647-293T cell group or Y1684-Y1647-293T cell group is used as a template.
  • a primer consisting of hRosa26-F: 5'-AACCTCGACACCAACTCTAGTCC-3' and hRosa26-R: 5'-TCTCACATGAGCGAAACCACTGC-3' was subjected to PCR amplification using Max enzyme to obtain a 670 bp PCR amplification product B.
  • the PCR amplification product A is subjected to gel recovery to obtain a recovered product A; the PCR amplification product B is subjected to gel recovery to obtain a recovered product B.
  • the annealing reaction system includes the recovered product A or the recovered product B 1-3 ⁇ g, the mutation detection buffer 3 ⁇ L, and the ddH 2 O is added to 30 ⁇ L.
  • step 2 After completing step 2, an annealing reaction is performed.
  • the reaction conditions were as follows: first at 98 ° C for 10 min, then slowly cooled (cooling rate ⁇ 1 ° C / 10 s) to 25 ° C, and finally 25 ° C for 5 min.
  • the annealing reaction system after completion of the step 3 is the prepared sample.
  • PCR amplification product A can be digested into two segments by T7EI and the size is about 198 bp and 277 bp, respectively, indicating that the corresponding CRISPR/Cpf1 system causes mutation of hAAVS1 gene; if the PCR amplification product A is not digested by T7EI Significantly changed, the corresponding CRISPR/Cpf1 system does not cause mutation of the hAAVS1 gene;
  • PCR product B can be digested into two segments by T7EI and the size is about 286 bp and 384 bp, respectively, indicating that the corresponding CRISPR/Cpf1 system causes the mutation of THUMPD3-AS1 gene; if the PCR amplification product B is digested by T7EI There was no significant change in size, and the corresponding CRISPR/Cpf1 system did not cause a mutation in the THUMPD3-AS1 gene.
  • FIG. 1 A is the hAVS1 gene mutation test in Figure 1: the upper left is the AsCRISPR/Cpf1 system, where M is DNA Marker, "-" is Y1681-293T cell group, and As is Y1681-Y1640- In the 293T cell group, Fn is the Y1681-Y1642-293T cell group, Lb is the Y1681-Y1644-293T cell group, Lb2 is the Y1681-Y1646-293T cell group, and the upper right is the FnCRISPR/Cpf1 system, where M is the DNA Marker, "-" For the Y1682-293T cell group, As is Y1682-Y1640-293T cell group, Fn is Y1682-Y1642-293T cell group, Lb is Y1682-Y1644-293T cell group, Lb2 is Y1682-Y1646-293T cell group; LbCRISPR is lower left.
  • B is the THUMPD3-AS1 gene.
  • the PCR amplification product A obtained in (2) in the step 2 was sequenced, and the primer was hAAV-ce: 5'-cagctcccccccccccttac-3'.
  • the PCR amplification product B obtained in (3) in the step 2 was subjected to sequencing, and the primer was hRosa26-ce: 5'-cgcccagggaccaagttagc-3'. The sequencing was completed by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • Figure 2 Figure 2
  • Figure 2 A is the editing ability of the AsCRISPR/Cpf1 system for the hAAVS1 gene, wherein (a) is the Y1681-293T cell group, (b) is the Y1681-Y1640-293T cell group, and (c) is Y1681.
  • B is the editing ability of the AsCRISPR/Cpf1 system for the THUMPD3-AS1 gene, (a) is the Y1681-293T cell group, (b) is the Y1681-Y1641-293T cell group, (c) is the Y1681-Y1643-293T cell group, (d) is the Y1681-Y1645-293T cell group, and (e) is In the Y1681-Y1647-293T cell group; in Figure 2, C is the editing ability of the FnCRISPR/Cpf1 system for the hAAVS1 gene, wherein (a) is the Y1682-293T cell group, and (b) is the Y1682-Y1640-293T cell group, (c) For the Y1682-Y1642-293T cell group, (d) is the
  • E is the LbCRISPR/Cpf1 system for hAAVS The editing ability of 1 gene, wherein (a) is Y1683-293T cell group, (b) is Y1683-Y1640-293T cell group, (c) is Y1683-Y1642-293T cell group, and (d) is Y1683-Y1644-293T In the cell group, (e) is the Y1683-Y1646-293T cell group; in Figure 2, F is the editing ability of the LbCRISPR/Cpf1 system for the THUMPD3-AS1 gene, wherein (a) is the Y1683-293T cell group, and (
  • H is the editing ability of the Lb2 CRISPR/Cpf1 system for the THUMPD3-AS1 gene, wherein (a) is the Y1684-293T cell group, and (b) is Y1684-Y1641-293T cell group, (c) is Y1684-Y1643-293T cell group, (d) is Y1684-Y1645-293T cell group, and (e) is Y1684-Y1647-293T cell group).
  • the crRNA shown in Table 1 was synthesized.
  • the Lb crRNA backbone sequence is underlined from the 5' end to the 2nd to 21st position
  • the dotted line is the Lb2crRNA backbone sequence from the 5' end to the 2nd to 20th position
  • the double underlined is the target sequence I from the 5' end.
  • the box is from position 1 to position 23 from the 5' end
  • the single wavy line is from position 1 to position 23 from the 5' end
  • the double wavy line is from target 5 to 5'. From the end to the 1st to 19th positions.
  • RNA solution having an RNA concentration of 0.5 ⁇ g/ ⁇ L.
  • Example 1 step 2 (1) is the same as in Example 1 step 2 (1).
  • Example 1 step 2 (1) is the same as in Example 1 step 2 (1).
  • the medium in the 6-well plate was changed to OPTI-MEM medium, and then 4 ⁇ g of plasmid Y1683 and 2 ⁇ g of A39 (ie, 4 ⁇ L of RNA solution) were added for co-transfection (in the process of co-transfection).
  • the transfection reagent is lipofectamine 2000
  • the medium is OPTI-MEM medium
  • the procedure of co-transfection is referenced to Lipofectamin2000 manual, and then incubated in 37 ° C, 5% CO 2 incubator for 6 h, and then replaced with new OPTI-MEM culture.
  • the cells were further cultured for 18 h in a 37 ° C, 5% CO 2 incubator.
  • Example 7 is the same as in Example 1 step 2 (1).
  • step 8 is the same as in step 1 of the first embodiment 1 (1).
  • Example 9 is the same as in Example 1 step 2 (1).
  • Example 1 step 2 (1) is the same as in Example 1 step 2 (1).
  • the precipitate in step 10 is the 293T cell group co-transfected with plasmid Y1683 and A39, referred to as Y1683-A39-293T cell group.
  • the plasmid A39 was replaced with R39, and the other steps were unchanged, and the Y1683-R39-293T cell group was obtained.
  • the plasmid Y1681 was replaced with plasmid Y1683 according to the method of (2) in the second step of Example 1, and the other steps were unchanged, and the Y1683-293T cell group was obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1644, and the plasmid Y1681 was replaced with the plasmid Y1683.
  • the other steps were unchanged, and the Y1683-Y1644-293T cell group was obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1645, and the plasmid Y1681 was replaced with the plasmid Y1683.
  • the other steps were unchanged, and the Y1683-Y1645-293T cell group was obtained.
  • the plasmid Y1683 was replaced with the plasmid Y1684, the plasmid A39 was replaced with A38, and the other steps were unchanged, and the Y1684-A38-293T cell group was obtained.
  • the plasmid Y1683 was replaced with the plasmid Y1684, the plasmid A39 was replaced with A42, and the other steps were unchanged, and the Y1684-A42-293T cell group was obtained.
  • the plasmid Y1683 was replaced with the plasmid Y1684, the plasmid A39 was replaced with R38, and the other steps were unchanged, and the Y1684-R38-293T cell group was obtained.
  • the plasmid Y1683 was replaced with the plasmid Y1684, the plasmid A39 was replaced with R42, and the other steps were unchanged, and the Y1684-R42-293T cell group was obtained.
  • Plasmid Y1681 was replaced with plasmid Y1684 according to the method of (2) in Example 1, Step 2, and the other steps were unchanged to obtain a Y1684-293T cell group.
  • the plasmid Y1640 was replaced with the plasmid Y1646, and the plasmid Y1681 was replaced with the plasmid Y1684.
  • the other steps were unchanged, and the Y1684-Y1646-293T cell group was obtained.
  • the plasmid Y1640 was replaced with the plasmid Y1647, and the plasmid Y1681 was replaced with the plasmid Y1684.
  • the other steps were unchanged, and the Y1684-Y1647-293T cell group was obtained.
  • the genomic DNA of each cell group obtained in the step 1 was separately extracted with a Genomic DNA Extraction kit.
  • step (1) Y1683-293T cell group, Y1683-Y1644-293T cell group, Y1684-Y1646-293T cell group, Y1683-A39-293T cell group, Y1684-A38 extracted by step (1)
  • the genomic DNA of the -293T cell group or the Y1684-A42-293T cell group was used as a template, and the primer consisting of hAAV-F: 5'-GGGTCACCTCTCACTCCTTTCAT-3' and hAAV-R: 5'-ATCCTCTCTGGCTCCATCGTAAG-3' was performed with Max enzyme.
  • PCR amplification revealed a 475 bp PCR amplification product A.
  • step (1) Y1684-293T cell group, Y1683-Y1645-293T cell group, Y1684-Y1647-293T cell group, Y1683-R39-293T cell group, Y1684-R38 extracted by step (1)
  • the genomic DNA of the -293T cell group or the Y1684-R42-293T cell group was used as a template, and the primer consisting of hRosa26-F: 5'-AACCTCGACACCAACTCTAGTCC-3' and hRosa26-R: 5'-TCTCACATGAGCGAAACCACTGC-3' was performed with Max enzyme.
  • PCR amplification revealed a 670 bp PCR amplification product B.
  • FIG. 3 A is a chemically synthesized crRNA used for mutation detection of hAAVS1 gene in CRISPR/Cpf1 system: M is DNA Marker, B is Y1683-293T cell group, and LbA is Y1683-Y1644-293T In the cell group, Lb2A is Y1684-Y1646-293T cell group, A39 is Y1683-A39-293T cell group, A38 is Y1684-A38-293T cell group, A42 is Y1684-A42-293T cell group; Figure 3 B is chemical synthesis.
  • the crRNA was used to detect the mutation of THUMPD3-AS1 gene in CRISPR/Cpf1 system: M is Marker, B is Y1684-293T cell group, LbR is Y1683-Y1645-293T cell group, Lb2R is Y1684-Y1647-293T cell group R39 is the Y1683-R39-293T cell group, R38 is the Y1684-R38-293T cell group, and R42 is the Y1684-R42-293T cell group).
  • M is Marker
  • B Y1684-293T cell group
  • LbR is Y1683-Y1645-293T cell group
  • Lb2R is Y1684-Y1647-293T cell group
  • R39 is the Y1683-R39-293T cell group
  • R38 is the Y1684-R38-293T cell group
  • R42 is the Y1684-R42-293T cell group
  • Figure 4 is the chemically synthesized crRNA used in the editing ability of hAAVS1 gene in CRISPR/Cpf1 system: LbCpf1 is Y1683-293T cell group, LbCpf1+LbA is Y1683-Y1644-293T cell group, Lb2Cpf1 +Lb2A is Y1684-Y1646-293T cell group, LbCpf1+A39 is Y1683-A39-293T cell group, Lb2Cpf1+A38 is Y1684-A38-293T cell group, and Lb2Cpf1+A42 is Y1684-A42-293T cell group.
  • Lb2Cpf1 is Y1684-293T cell group
  • LbCpf1+LbR is Y1683-Y1645-293T cell group
  • Lb2Cpf1+Lb2R is Y1684-Y1647- 293T cell group
  • LbCpf1+R39 is the Y1683-R39-293T cell group
  • Lb2Cpf1+R38 is the Y1684-R38-293T cell group
  • Lb2Cpf1+R42 is the Y1684-R42-293T cell group).
  • the sequencing results showed that the chemically synthesized crRNA can effectively perform the function of guiding Cpf1 to cleave and edit specific target sites, and the efficiency is high. Therefore, the chemically synthesized crRNA has certain gene editing ability in both the LbCRISPR/Cpf1 system and the Lb2 CRISPR/Cpf1 system.
  • the hAAVS1 gene (Gene ID: 54776) and the THUMPD3-AS1 gene (Gene ID: 440944) were selected as target genes for detecting the gene editing ability of the CRISPR/Cpf1 system.
  • the CRISPR/Cpf1 system is specifically an AsCRISPR/Cpf1 system or an FnCRISPR/Cpf1 system.
  • the AsCRISPR/Cpf1 system is derived from Acidaminococcus_sp.BV3L6, which expresses the AsCpf1 protein shown in SEQ ID NO:6 in the Sequence Listing.
  • the FnCRISPR/Cpf1 system is from Francisella_novicida, which expresses the FnCpf1 protein shown in SEQ ID NO: 5 in the Sequence Listing.
  • target sequence I is selected based on the nucleotide sequence of the hAAVS1 gene
  • target sequence II is selected based on the nucleotide sequence of the THUMPD3-AS1 gene.
  • sequences of target sequence I and target sequence II are as follows:
  • Target sequence I 5'-TCTGTCCCCTCCACCCCACAGTGG-3';
  • Target sequence II 5'-GAGAACAAGCGCCTCCCACCCACA-3'.
  • step 1 of the first embodiment Same as in step 1 of the first embodiment.
  • thymidine deoxynucleotide modification refers to the addition of 1-2 thymidine deoxynucleotides at the 5' and/or 3' end of the crRNA, represented by dT in the table.
  • the underlined is the As crRNA backbone sequence from the 5' end to the 2nd to 20th position
  • the dotted line is the Fn crRNA backbone sequence from the 5' end from the 2nd to the 20th position
  • the double underlined is the target sequence I from the 5' end. From positions 1 to 21, the box is from position 1 to position 21 of the target sequence II from the 5' end.
  • RNA solution having a concentration of 0.5 ⁇ g/ ⁇ L.
  • step 2 After completing step 1, add 1 mL of Trypsin-EDTA Solution to the culture dish, mix, then aspirate the liquid phase, and let stand at 37 ° C for 1-2 min.
  • step 2 2 mL of DMEM medium containing 10% by volume of FBS was added to the culture dish, and blown to form a single cell suspension.
  • step 3 the single cell suspension was inoculated into a 6-well plate, and about 2 ⁇ 10 5 293T cells were inoculated into each well, and cultured in a 37° C., 5% CO 2 incubator for 24 hours.
  • the medium in the 6-well plate was changed to OPTI-MEM medium, and then 4 ⁇ g of plasmid Y1681 and 4 ⁇ g of plasmid Y1640 were added for co-transfection (in the process of co-transfection, the transfection reagent was lipofectamine). 2000, the medium is OPTI-MEM medium, the process of co-transfection is referred to Lipofectamin2000 manual), then incubated in 37 ° C, 5% CO 2 incubator for 6 h, then replaced with new OPTI-MEM medium, 37 ° C, 5 Incubation was continued for 18 h in a %CO 2 incubator.
  • step 7 After 48 hours from the completion of step 6, the cells were collected and washed once with 1 mL of PBS buffer.
  • step 7 0.5 mL of Trypsin-EDTA Solution was added to the culture dish, mixed, and then the liquid phase was aspirated, and allowed to stand at 37 ° C for 1 to 2 minutes.
  • step 8 After completion of step 8, 1 mL of DMEM medium containing 10% by volume of FBS was added to the culture dish, and a single cell suspension was formed by pipetting; the single cell suspension was centrifuged at 1000 rpm for 3 minutes to obtain a precipitate 1.
  • Precipitate 2 is the 293T cell group co-transfected with plasmid Y1681 and plasmid Y1640, referred to as Y1681-Y1640-293T cell group.
  • the plasmid Y1640 was replaced with the plasmid Y1641, and the other steps were unchanged, and the Y1681-Y1641-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the plasmid Y1640 was replaced with the plasmid Y1642, and the other steps were unchanged, and the Y1682-Y1642-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the plasmid Y1640 was replaced with the plasmid Y1643.
  • the other steps were unchanged, and the Y1682-Y1643-293T cell group was obtained.
  • the medium in the 6-well plate was changed to OPTI-MEM medium, and then 4 ⁇ g of plasmid Y1681 was added for transfection (in the process of co-transfection, the transfection reagent was lipofectamine 2000, and the medium was OPTI-MEM medium, co-transfection step reference Lipofectamin2000 instructions), then incubated in 37 ° C, 5% CO 2 incubator for 6h, then replaced with new OPTI-MEM medium, 37 ° C, 5% CO 2 incubator Continue to culture for 18h.
  • the precipitate obtained in step 10 is the 293T cell group transfected with plasmid Y1681, referred to as Y1681-293T cell group.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the other steps were unchanged, and the Y1682-293T cell group was obtained.
  • the medium in the 6-well plate was changed to OPTI-MEM medium, and then 4 ⁇ g of plasmid Y1681 and 2 ⁇ g of A1 (ie, 4 ⁇ L) were added for co-transfection (co-transfection, transfection)
  • the reagent is lipofectamine 2000
  • the medium is OPTI-MEM medium
  • the procedure of co-transfection is referred to Lipofectamin2000 manual
  • incubated in 37 ° C, 5% CO 2 incubator for 6 h and then replaced with new OPTI-MEM medium, 37 Incubation was continued for 18 h in a °C, 5% CO 2 incubator.
  • the precipitate obtained in step 10 is the 293T cells co-transfected with plasmid Y1683 and A1, referred to as Y1683-A1-293T cell group.
  • step (7) A1 was replaced with A3, and the other steps were unchanged, and the Y1681-A3-293T cell group was obtained.
  • step (7) A1 was replaced with A5, and the other steps were unchanged, and the Y1681-A5-293T cell group was obtained.
  • step (7) A1 was replaced with A7, and the other steps were unchanged, and the Y1681-A7-293T cell group was obtained.
  • step (7) A1 was replaced with A9, and the other steps were unchanged, and the Y1681-A9-293T cell group was obtained.
  • step (7) A1 was replaced with R1, and the other steps were unchanged, and the Y1681-R1-293T cell group was obtained.
  • step (7) A1 was replaced with R3, and the other steps were unchanged, and the Y1681-R3-293T cell group was obtained.
  • step (7) A1 was replaced with R5, and the other steps were unchanged, and the Y1681-R5-293T cell group was obtained.
  • step (7) A1 was replaced with R7, and the other steps were unchanged, and the Y1681-R7-293T cell group was obtained.
  • step (7) A1 was replaced with R9, and the other steps were unchanged, and the Y1681-R9-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the A1 was replaced with A2, and the other steps were unchanged, and the Y1682-A2-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the A1 was replaced with A4, and the other steps were unchanged, and the Y1682-A4-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the A1 was replaced with A6, and the other steps were unchanged, and the Y1682-A6-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the A1 was replaced with A8, and the other steps were unchanged, and the Y1682-A8-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the A1 was replaced with A10, and the other steps were unchanged, and the Y1682-A10-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the A1 was replaced with R2.
  • the other steps were unchanged, and the Y1682-R2-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the A1 was replaced with R4, and the other steps were unchanged, and the Y1682-R4-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the A1 was replaced with R6, and the other steps were unchanged, and the Y1682-R6-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the A1 was replaced with R8, and the other steps were unchanged, and the Y1682-R8-293T cell group was obtained.
  • the plasmid Y1681 was replaced with the plasmid Y1682, and the A1 was replaced with R10, and the other steps were unchanged, and the Y1682-R10-293T cell group was obtained.
  • the genomic DNA of each cell group obtained in the step 1 was separately extracted with a Genomic DNA Extraction kit.
  • step (1) Y1682-Y1642-293T cell group, Y1682-293T cell group, Y1682-Y1643-293T cell group, Y1682-A2-293T cell group, Y1682-A4 extracted by step (1) -293T cell group, Y1682-A6-293T cell group, Y1682-A8-293T cell group, Y1682-A10-293T cell group, Y1682-R2-293T cell group, Y1682-R4-293T cell group, Y1682-R6-293T Genomic DNA of the cell group, Y1682-R8-293T cell group or Y1682-R10-293T cell group is a template, consisting of hRosa26-F: 5'-AACCTCGACACCAACTCTAGTCC-3' and hRosa26-R: 5'-TCTCACATGAGCGAAACCACTGC-3' The primer was subjected to PCR amplification using Max enzyme to obtain a 670 bp PCR amplification
  • the PCR amplification product A is subjected to gel recovery to obtain a recovered product A; the PCR amplification product B is subjected to gel recovery to obtain a recovered product B.
  • an annealing reaction system is prepared: the annealing reaction system comprises recovering product A or 500 ng of recovered product, 2 ⁇ L of mutation detection buffer, and supplementing to 20 ⁇ L with ddH 2 O.
  • an annealing reaction is performed. The reaction conditions were as follows: first at 98 ° C for 10 min, then slowly cooled (cooling rate ⁇ 1 ° C / 10 s) to 25 ° C, and finally 25 ° C for 5 min.
  • the annealing reaction system after completion of the step 3 is the prepared sample.
  • PCR amplification product A can be digested into two segments by T7EI and the size is about 198 bp and 277 bp, respectively, indicating that the corresponding CRISPR/Cpf1 system causes mutation of hAAVS1 gene; if the PCR amplification product A is not digested by T7EI Significantly changed, the corresponding CRISPR/Cpf1 system does not cause mutation of the hAAVS1 gene;
  • PCR product B can be digested into two segments by T7EI and the size is about 286 bp and 384 bp, respectively, indicating that the corresponding CRISPR/Cpf1 system causes the mutation of THUMPD3-AS1 gene; if the PCR amplification product B is digested by T7EI There was no significant change in size, and the corresponding CRISPR/Cpf1 system did not cause a mutation in the THUMPD3-AS1 gene.
  • T7E1 mutation assay The results of T7E1 mutation assay are shown in Figure 5.
  • M DNA Marker
  • "-" is Y1681-293T cell group
  • "+” is Y1681-Y1640-293T.
  • A1 is Y1681-A1-293T cell group
  • A3 is Y1681-A3-293T cell group
  • A5 is Y1681-A5-293T cell group
  • A7 is Y1681-A7-293T cell group
  • A9 is Y1681-A9-293T.
  • the cell group; the lower left panel shows the results of mutation detection of the THUMPD3-AS1 gene in the AsCRISPR/Cpf1 system, wherein M is DNA Marker, "-" is Y1681-293T cell group, and “+” is Y1681-Y1641-293T cell group, R1 For the Y1681-R1-293T cell group, R3 is the Y1681-R3-293T cell group, R5 is the Y1681-R5-293T cell group, R7 is the Y1681-R7-293T cell group, and R9 is the Y1681-R9-293T cell group;
  • the picture shows the mutation detection of hAAVS1 gene in FnCRISPR/Cpf1 system, wherein M is DNA Marker, "-” is Y1682-293T cell group, "+” is Y1682-Y1642-293T cell group, A2 is Y1682-A2-293T In the cell group, A4 is the Y1682-A4-293T
  • R4 is the Y1682-R4-293T cell group
  • R6 is the Y1682-R6-293T cell group
  • R8 is the Y1682-R8-293T cell group
  • R10 is the Y1682-R10-293T cell group).
  • the PCR amplification product A obtained in (2) of step 2 was sequenced, and the primer was hAAV-ce: 5'-cagctcccctaccccccttac-3'.
  • the PCR amplification product B obtained in (3) in the step 2 was subjected to sequencing, and the primer was hRosa26-ce: 5'-cgcccagggaccaagttagc-3'. The sequencing was completed by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • A is the sequencing result of hAAVS1 gene in AsCRISPR/Cpf1 system, wherein AsCpf1 is Y1681-293T cell group, AsCpf1+Plasmid AAVS1crRNA is Y1681-Y1640-293T cell group, and AsCpf1+A1 is Y1681-A1-293T In the cell group, AsCpf1+A3 was Y1681-A3-293T cell group, AsCpf1+A5 was Y1681-A5-293T cell group, AsCpf1+A7 was Y1681-A7-293T cell group, and AsCpf1+A9 was Y1681-A9-293T cell group.
  • B is the sequencing result of hNAVS1 gene of FnCRISPR/Cpf1 system, wherein AsCpf1 is Y1682-293T cell group, FnCpf1+Plasmid AAVS1crRNA is Y1682-Y1642-293T cell group, FnCpf1+A2 is Y1682-A2-293T cell group, FnCpf1+ A4 is Y1682-A4-293T cell group, FnCpf1+A6 is Y1682-A6-293T cell group, FnCpf1+A8 is Y1682-A8-293T cell group, FnCpf1+A10 is Y1682-A10-293T cell group;
  • C is AsCRISPR/ The sequencing results of THUMPD3-AS1 gene in Cpf1 system, wherein AsCpf1 is Y1681-293T cell group, AsCpf1+Plasmid THUMPD3AS1crRNA is Y1681-Y
  • AsCpf1+R9 is the Y1681-R9-293T cell group; D is the sequencing result of THUMPD3-AS1 gene in FnCRISPR/Cpf1 system, FnCpf1 is Y1682-293T cell group, FnCpf1+Plasmid THUMPD3Fn1crRNA is Y1682-Y1643-293T cell group, FnCpf1 +R2 is Y1682-R2-293T cell group, FnCpf1+R4 is Y1682-R4-293T cell group, FnCpf1+R6 is Y1682-R6-293T cell group, FnCpf1+R8 is Y1682-R8-293T cell group, FnCpf1+R10 For the Y1682-R10-293T cell group).
  • U-methoxy modification refers to the addition of a methoxy-modified uridine ribonucleotide at the 5' and/or 3' end of the crRNA, expressed as mU in the table
  • U-thio Modification refers to the addition of a thio-modified uridine ribonucleotide at the 5' and/or 3' end of the crRNA, indicated by *U in the table
  • U-methoxythio modification refers to Addition of a methoxythio-modified uridine ribonucleotide to the 5' and/or 3' end of the crRNA, indicated by *mU in the table
  • UF modification refers to the 5' end of the crRNA and/or Addition of a 1 F-modified uridine ribonucleotide at the 3' end, indicated by fU in the table
  • U-methoxy modification refers to the addition of a methoxy-modified uridine ribonucleo
  • the vector-expressed crRNA has certain gene editing ability in the AsCRISPR/Cpf1 system, the FnCRISPR/Cpf1 system, the LbCRISPR/Cpf1 system, and the Lb2 CRISPR/Cpf1 system.
  • the chemically synthesized crRNA can be directly transfected, is easier to manipulate, more controllable, and facilitates chemical modification than transfection by constructing a recombinant vector.
  • the chemically synthesized and modified crRNA has a stronger gene editing ability than the chemically synthesized crRNA. Therefore, vector-expressed crRNA and/or chemically synthesized crRNA and/or chemically synthesized and modified crRNA for the CRISPR/Cpf1 system have important application value in gene editing.

Abstract

La présente invention repose sur l'utilisation du système CRISPR/Cpf1 dans l'édition de gènes, les éléments a1) ou a2) ou a3) suivants étant inclus dans le système CRISPR/Cpf1 : a1) un ARNcr obtenu par chimiosynthèse ; a2) un ARNcr obtenu par chimiosynthèse et modifié ; et a3) un vecteur exprimant un ARNcr. Le procédé de modification comprend la modification d'un acide désoxyribonucléique. L'acide désoxyribonucléique est un désoxynucléotide thymidine. Des expériences montrent que l'ARNcr obtenu par chimiosynthèse et l'ARNcr obtenu par chimiosynthèse et modifié pour le système CRISPR/Cpf1 entraînent tous les deux des mutations dans le gène hAAVS1 et le gène THUMPD3-AS1, indiquant ainsi qu'ils présentent tous les deux une certaine capacité d'édition de gènes. L'ARNcr exprimé par un vecteur, l'ARNcr obtenu par chimiosynthèse et l'ARNcr obtenu par chimiosynthèse et modifié pour le système CRISPR/Cpf1 présentent une valeur d'application importante dans l'édition de gènes.
PCT/CN2017/082968 2016-05-05 2017-05-04 Utilisation d'arncr et d'arncr modifié obtenus par chimiosynthèse dans des systèmes d'édition de gènes crispr/cpf1 WO2017190664A1 (fr)

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CN201610703716.7A CN106244591A (zh) 2016-08-23 2016-08-23 修饰crRNA在CRISPR/Cpf1基因编辑系统中的应用

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