[go: up one dir, main page]

WO2018177351A1 - Procédé de préparation d'un animal à invalidation génique non chimérique basé sur la technologie crispr/cas9 - Google Patents

Procédé de préparation d'un animal à invalidation génique non chimérique basé sur la technologie crispr/cas9 Download PDF

Info

Publication number
WO2018177351A1
WO2018177351A1 PCT/CN2018/081016 CN2018081016W WO2018177351A1 WO 2018177351 A1 WO2018177351 A1 WO 2018177351A1 CN 2018081016 W CN2018081016 W CN 2018081016W WO 2018177351 A1 WO2018177351 A1 WO 2018177351A1
Authority
WO
WIPO (PCT)
Prior art keywords
sgrna
seq
gene
target
target gene
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2018/081016
Other languages
English (en)
Chinese (zh)
Inventor
杨辉
熊志奇
孙强
左二伟
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Institutes for Biological Sciences SIBS of CAS
Original Assignee
Shanghai Institutes for Biological Sciences SIBS of CAS
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 Shanghai Institutes for Biological Sciences SIBS of CAS filed Critical Shanghai Institutes for Biological Sciences SIBS of CAS
Publication of WO2018177351A1 publication Critical patent/WO2018177351A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • C12N15/87—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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • 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
    • 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
    • C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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

Definitions

  • the present invention belongs to the field of molecular biology, and more particularly, to a method based on CRISPR/Cas9 technology, which is a modified first-generation gene to completely knock out or most knock out a gene knockout animal. .
  • CRISPR/Cas9 is derived from the immune system of bacteria and archaea, and can be used to bring Cas9 nuclease to a specific location on the genome using target-specific RNA to achieve precise cleavage of the gene locus.
  • CRISPR/Cas9 technology has been applied to the establishment of disease models, drug target screening, and is becoming a new generation of gene therapy.
  • the CRISPR/Cas9 system has been used for genome editing in many species. It is now possible to directly inject Cas9 mRNA and sgRNA (single-guide RNA) into the pronuclear fertilized egg to introduce a double-strand break at a specific position (double-strand break) , DSB).
  • the double-strand break introduced by CRISPR/Cas9 is treated by a dislocation repair mechanism called non-homologous end joining (NHEJ), which causes the mutant animal to carry a frameshift mutation.
  • NHEJ non-homologous end joining
  • most of the mutant animals obtained by this method have a chimeric phenomenon, that is to say, their genetic mutations are only present in some cells and other cells are not.
  • a method of producing a knock-out animal cell comprising: (1) preparing two or more targeting target genes differently depending on a nucleic acid sequence of a target gene to be knocked out The sgRNA of the target site; (2) co-transfecting the sgRNA of (1) or the nucleic acid capable of forming the sgRNA, Cas9 mRNA or a nucleic acid capable of forming the Cas9 mRNA into an animal cell to obtain a knock-out animal cell.
  • the animal cell is an animal fertilized egg
  • the fertilized egg can develop into a knockout animal in which the gene function of the target gene is completely knocked out or mostly knocked out.
  • a method of producing a gene knock-out or a majority knock-out gene knockout animal of a gene of a target gene comprising: (1) depending on a nucleic acid sequence of a target gene to be knocked out, Preparing two or more sgRNAs targeting different target sites on the target gene; (2) co-transferring the sgRNA of (1) or the nucleic acid capable of forming the sgRNA, Cas9 mRNA or nucleic acid capable of forming the Cas9 mRNA
  • the fertilized egg of the gene knockout animal is obtained; (3) the fertilized egg of (2) is developed to produce a gene knockout animal in which the gene function of the target gene is completely knocked out or most knocked out.
  • the two or more sgRNAs targeting different target sites on the target gene are between 9 and 500 bp apart from the target sites on the target gene of interest; preferably 10 to 300 bp, for example, 15 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp, 150 bp, 180 bp, 200 bp, 250 bp, 280 bp.
  • a plurality of sgRNAs targeting different target sites on the target gene are used; for example, 3 to 30, for example, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18 , 20, 25 articles.
  • the plurality of sgRNAs targeting different target sites on the target gene at least 2 (preferably at least 3, for example, 4, 5, 6, 7, 8, 9 10, 12, 15, 18, 20, 25)
  • the target site on the target gene targeted is located in the same exon region.
  • the plurality of sgRNAs targeting different target sites on the target gene introduce a frameshift mutation and/or introduce an insertion deletion and/or a target site region on the target gene of interest. Introducing large fragment deletions.
  • the nucleic acid capable of forming the sgRNA is a nucleic acid construct or an expression vector
  • the nucleic acid capable of forming the Cas9 mRNA is a nucleic acid construct or an expression vector.
  • the animal is a mammal, including but not limited to: human, non-human primate, mouse, domestic animal.
  • the nucleic acid sequence of the sgRNA carries a promoter upstream, preferably the promoter is a T7 promoter, a U6 promoter; or the upstream of the nucleic acid sequence of the Cas9 mRNA carries a promoter, Preferably the promoter is a T7 promoter.
  • the gene function of the target gene is mostly knocked out in the body of the knockout animal, and the cell to be knocked out of the target gene does not effectively knock out less than 20% of the total number of cells; more preferably 15% or less; more preferably 10% or less; further preferably 5% or less.
  • the target gene to be knocked out is a GFP gene
  • the sgRNA is 2, 3 or 4
  • the interval between target sites on each target gene targeted by each sgRNA is 30 ⁇ . 200 bp; preferably 40 to 150 bp; more preferably 50 to 130 bp; more preferably, the target site on the target gene targeted by the sgRNA is 2, 3 or 4 selected from the group consisting of SEQ ID NO: :48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51.
  • the target gene to be knocked out is a Tyr gene
  • the sgRNA is 3, 4, 5 or 6, and the exon 4 of the target gene targeted by each sgRNA and optionally An intron adjacent to the exon
  • the interval between the target sites targeted by the sgRNA is 10 to 100 bp; preferably 10 to 70 bp; more preferably, the target of the target gene targeted by the sgRNA
  • the site is 3, 4, 5 or 6 selected from the group consisting of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO More preferably: 3 or 4 selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56.
  • the target gene to be knocked out is a Tet1 gene
  • the sgRNA is 3, the exon 2 of the target gene targeted by each sgRNA, and the target site targeted by each sgRNA
  • the interval between 80 and 200 bp; preferably 100 to 190 bp; more preferably, the target site on the target gene targeted by the sgRNA is: SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO :60.
  • the target gene to be knocked out is a Tet2 gene
  • the sgRNA is 3, the exon 3 of the target gene targeted by each sgRNA, and the target site targeted by each sgRNA
  • the interval between 100 and 150 bp; preferably 110 to 135 bp; more preferably, the target site on the target gene targeted by the sgRNA is: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO :63.
  • the target gene to be knocked out is a Tet3 gene
  • the sgRNA is 3, the exon 4 of the target gene targeted by each sgRNA, and the target site targeted by each sgRNA
  • the interval between 180 and 280 bp; preferably 190 to 270 bp; more preferably, the target site on the target gene targeted by the sgRNA is: SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO :66.
  • the target gene to be knocked out is a Prrt2 gene
  • the sgRNA is 3 or 4, at least 3 exons 3 of the target gene targeted by the sgRNA, and the exon 3
  • the interval between the target sites targeted by the sgRNA is 10 to 50 bp; preferably 15 to 40 bp; more preferably, the target site on the target gene targeted by the sgRNA is: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86; more preferably: SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86.
  • the target gene to be knocked out is an ArntL gene
  • the sgRNA is 3, 4, 5, 6, 7, 8, or 9 and the exon 13 of the targeted target gene is targeted.
  • the interval between the target sites targeted by the sgRNA on the exon 13 is 10 to 80 bp; preferably 40 to 70 bp; more preferably, the target site on the target gene targeted by the sgRNA is selected from the group consisting of 3, 4, 5 or 6 of the next group: SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99; more preferably: SEQ ID NO: 92, SEQ ID NO: 95, SEQ ID NO: 98.
  • the target gene to be knocked out is a Y chromosome gene; preferably Zfy1; Ube1y1; Kdm5d; Eif2s3y; Ddx3y; Usp9y; Sry; Erdr1;
  • the target gene to be knocked out is Zfy1
  • the target site on the target gene targeted by the sgRNA is SEQ ID NO: 67, SEQ ID NO: 68;
  • the target gene to be knocked out is Ube1y1, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 69, SEQ ID NO: 70;
  • the target gene to be knocked out is Kdm5d, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 71, SEQ ID NO: 72;
  • the target gene to be knocked out is Eif2s3y, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 73, SEQ ID NO: 74;
  • the target gene to be knocked out is Ddx3y, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 75, SEQ ID NO: 76;
  • the target gene to be knocked out is Usp9y, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 77, SEQ ID NO: 78;
  • the target gene to be knocked out is Sry, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 79, SEQ ID NO: 80;
  • the target gene to be knocked out is Erdr1, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 81, SEQ ID NO: 82.
  • the animal whose function of the target gene is completely knocked out or largely knocked out is an animal obtained only after one birth cycle (i.e., an animal of the F0 generation).
  • it is also used for performing animal gene function research; or for preparing a target gene whose function is completely knocked out or most knocked out, and using it for gene function research, or using it for Embryonic development research.
  • the use is for non-diagnostic or therapeutic use.
  • a sgRNA of a gene knockout animal or a nucleic acid capable of forming the sgRNA which is a full knockout or a majority knockout of a gene for producing a target gene, the sgRNA being used for knocking a target base
  • the sgRNA is 2, 3 or 4
  • the distance between the target sites on the target gene targeted by each sgRNA is 30-200 bp; preferably 40-150 bp; more preferably 50-130 bp; More preferably, the target site on the target gene targeted by the sgRNA is 2, 3 or 4 selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51.
  • a sgRNA of a gene knockout animal or a nucleic acid capable of forming the sgRNA which is a full knockout or a majority knockout of a gene for producing a target gene, the sgRNA being used for knocking a target base
  • the sgRNA is 3, 4, 5 or 6, the exon 4 of the target gene targeted by each sgRNA and optionally an intron adjacent to the exon, and the sgRNA is targeted
  • the interval between the target sites is 10 to 100 bp; preferably 10 to 70 bp; more preferably, the target site on the target gene targeted by the sgRNA is 3, 4, 5 or 6 selected from the group below SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57; more preferably 3 or 4 selected from the group consisting of SEQ ID NO:53, SEQ ID NO:54, SEQ
  • a sgRNA of a gene knockout animal or a nucleic acid capable of forming the sgRNA which is a full knockout or a majority knockout of a gene for producing a target gene, the sgRNA being used for knocking a target base
  • the sgRNA is 3, the exon 2 of the target gene targeted by each sgRNA, and the interval between the target sites targeted by each sgRNA is 80-200 bp; preferably 100-190 bp;
  • the target site on the target gene targeted by the sgRNA is: SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60.
  • a sgRNA of a gene knockout animal or a nucleic acid capable of forming the sgRNA which is a full knockout or a majority knockout of a gene for producing a target gene, the sgRNA being used for knocking a target base
  • the sgRNA is three, the exon 3 of the target gene targeted by each sgRNA, and the interval between the target sites targeted by each sgRNA is 100-150 bp; preferably 110-135 bp;
  • the target site on the target gene targeted by the sgRNA is: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63.
  • a sgRNA of a gene knockout animal or a nucleic acid capable of forming the sgRNA which is a full knockout or a majority knockout of a gene for producing a target gene, the sgRNA being used for knocking a target base
  • the sgRNA is 3, the exon 4 of the target gene targeted by each sgRNA, and the interval between the target sites targeted by each sgRNA is 180-280 bp; preferably 190-270 bp;
  • the target site on the target gene targeted by the sgRNA is: SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66.
  • a sgRNA of a gene knockout animal or a nucleic acid capable of forming the sgRNA which is a full knockout or a majority knockout of a gene for producing a target gene, the sgRNA being used for knocking a target base
  • the sgRNA is 3 or 4
  • the target site on the target gene targeted by the sgRNA is: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 More preferably: SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86.
  • a sgRNA capable of completely knocking out a gene of a target gene or a majority of a knockout gene knockout animal or a nucleic acid capable of forming the sgRNA
  • the target gene for knocking out the sgRNA is The ArntL gene
  • the sgRNA is 3, 4, 5, 6, 7, 8, or 9, targeting the exon 13 of the target gene, and the interval between the target sites targeted by the sgRNA on exon 13 Is 10 to 80 bp; preferably 40 to 70 bp; more preferably, the target site on the target gene targeted by the sgRNA is 3, 4, 5 or 6 selected from the group consisting of SEQ ID NO: 91 SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99;
  • a sgRNA of a gene knockout animal or a nucleic acid capable of forming the sgRNA which is a full knockout or a majority knockout of a gene for producing a target gene, the sgRNA being used for knocking a target base
  • the target site on the target gene targeted by the sgRNA is SEQ ID NO: 67, SEQ ID NO: 68;
  • the target gene for knocking out the sgRNA is the Y chromosome gene Ube1y1, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 69, SEQ ID NO: 70;
  • the target gene for knocking out the sgRNA is the Y chromosome gene Kdm5d, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 71, SEQ ID NO: 72;
  • the target gene for knocking out the sgRNA is the Y chromosome gene Eif2s3y, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 73, SEQ ID NO: 74;
  • the target gene for knocking out the sgRNA is the Y chromosome gene Ddx3y, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 75, SEQ ID NO: 76;
  • the target gene for knocking out the sgRNA is the Y chromosome gene Usp9y, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 77, SEQ ID NO: 78;
  • the target gene for knocking out the sgRNA is the Y chromosome gene Sry, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 79, SEQ ID NO: 80;
  • the target gene for knocking out the sgRNA is the Y chromosome gene Erdr1, and the target site on the target gene targeted by the sgRNA is SEQ ID NO: 81, SEQ ID NO: 82.
  • kits for preparing a gene knockout or a majority knockout gene knockout animal of a gene of interest comprising any of the foregoing for preparation of a target
  • the gene function of the gene completely knocks out the sgRNA of the most knocked out animal, or the nucleic acid capable of forming the sgRNA.
  • the kit further comprises: Cas9 mRNA or a nucleic acid capable of forming the Cas9 mRNA; or instructions for use.
  • FIG. 1 Complete knockout of the GFP gene in GFP embryos with C-CRISPR.
  • FIG. 1 Schematic representation of the sgRNA target and experimental design at the GFP site.
  • Cas9 mRNA and single or multiple GFP-targeting sgRNAs were co-injected into individual mouse fertilized eggs and their GFP signal was observed at the blastocyst stage.
  • GFP IF and GFP IR internal primers identified by GFP genotype; external primers identified by GFP OF and GFP OR: GFP genotypes.
  • GFP IF and GFP IR internal primers identified by GFP genotype
  • external primers identified by GFP OF and GFP OR GFP genotypes.
  • x which is a specific number
  • GFP-negative blastocysts green arrows, no GFP signal in any cells in the blastocyst
  • GFP-positive chimeric blastocysts red arrows, some cells in the blastocyst have GFP signals.
  • (C) A histogram of the proportion of GFP-negative blastocysts (no chimerism) produced by various GFP-targeted methods. Two or more sgRNA targeting (sgRNA-GFP-A+B, A+B+C or A+B+C+D) than single sgRNA targeting (sgRNA-GFP-A, B, C, D) A higher proportion of GFP-negative blastocysts is produced. The number on the bar represents the total number of blastocysts counted (***P ⁇ 0.001, chi-square test).
  • Figure 2 One-step method for obtaining non-chimeric Tyr knockout mice using C-CRISPR.
  • Cas9 mRNA is co-injected with single or multiple sgRNAs that target Tyr into a single mouse fertilized egg and then transplanted to the recipient.
  • Tyr IF and Tyr IR internal primers identified by the Tyr genotype;
  • Tyr OF and Tyr OR external primers identified by the Tyr genotype.
  • (D) A histogram of the proportion of albino mice targeted by Tyr.
  • Two or more sgRNA targets sgRNA-Tyr-B+C, B+C+D, B+C+D+E, A+C+F or A+C+D+F
  • sgRNA-Tyr-C or D The numbers represent the total number of mice counted (***P ⁇ 0.001, chi-square test).
  • mice The tail of an albino mouse targeted by sgRNA-Tyr-B+C+D+E was sequenced and identified. #2 mice have large fragment exon deletions and insertion deletions. LED, large fragment exon deletion; Indel, insert or delete base. Number above each column: total number of TA clones sequenced.
  • E&F Representative sequence of a single blastomere in a 16-cell embryo produced by sgRNA-Tyr-C (E) and sgRNA-Tyr-B+C+D+E(F). Approximately 50% of blastomeres per embryo are successfully amplified and sequenced. Sequences targeted by sgRNA are marked in green, while PAM sequences are marked in red; deleted nucleotides are indicated by hyphens, and dashed lines indicate areas that are omitted.
  • G&H Tyr targets the proportion of different mutation types (G) and genotypes (H) of individual blastomeres of 8 to 16 cell embryos.
  • LED large fragment exon deletion
  • Bi-allelic biallelic mutation
  • Mono-allelic single allelic mutation.
  • the numbers represent the total number of alleles or blastomeres analyzed.
  • Figure 4 One-step method for obtaining a chimeric triple Tet knockout mouse using C-CRISPR.
  • mice obtained by knocking out a single Y chromosome gene by the C-CRISPR method.
  • the numbers represent the total number of transplanted embryos. Targeting the Erdr1 gene results in embryonic lethality.
  • a PCR product including a targeting site was amplified from a single blastomere of an sgRNA-Prrt2-C or sgRNA-Prrt2-B+C+D-targeted eight-cell embryo and subjected to Sanger sequencing. Sequences targeted by sgRNA are marked in green, while PAM sequences are marked in red; deleted nucleotides are indicated by hyphens, and dashed lines indicate areas that are omitted.
  • Prrt2 targets the ratio of different mutation types (C) and genotype (D) of a single blastomere of an eight-cell embryo obtained. LED, large fragment exon deletion.
  • Prrt2 targets the proportion of different mutation types in the tail, ears and blood cells of aborted and surviving monkeys.
  • Abortion monkeys were obtained by sgRNA-Prrt2-A targeting: monkeys #2, #3; surviving monkeys were obtained by sgRNA-Prrt2-A targeting: monkeys #4, #8, #10; by sgRNA-Prrt2-B+C+ D targeted to obtain surviving monkeys: monkey #11, #12.
  • Figure 7 Mouse chimeric rate obtained after co-injection of Cas9 mRNA and sgRNA into mouse MII oocytes.
  • Tet1 or Tet2IF and IR internal primers identified by the Tet1, Tet2 genotype; Tet1 or Tet2OF and OR: external primers identified by the Tet1, Tet2 genotype.
  • (E) A histogram of the proportion of chimeric blastocysts obtained by the one-step method and the two-step method, which were determined based on the results of DNA sequencing. The number on the line indicates the total number of blastocysts sequenced.
  • Figure 8 Representative PCR products and sequences of mouse tails, blastocysts and cleavage obtained by targeting gene Tyr.
  • sgRNA-Tyr-B+C+D+E targeted to obtain a representative sequence of mouse #5 tail. Sequences targeted by sgRNA are marked in green, while PAM sequences are marked in red; deleted nucleotides are indicated by hyphens, and dashed lines indicate areas that are omitted.
  • sgRNA-Tyr-C or sgRNA-Tyr-B+C+D+E is targeted to obtain representative PCR products of blastomeres.
  • the targeting gene Tyr obtains the proportion of blastomeres successfully amplified in the embryo.
  • the numbers represent the total number of blastomeres isolated from embryos obtained from the targeted gene Tyr.
  • D&E Tyr targets the ratio of different mutation types (G) and genotype (H) of a single blastomere of a two-cell embryo.
  • LED large fragment exon deletion
  • Bi-allelic biallelic mutation
  • Mono-allelic single allelic mutation.
  • the numbers represent the total number of alleles or blastomeres analyzed.
  • F&G sgRNA-Tyr-C
  • G Targeted sequence of a single blastomere in a two-cell embryo obtained. Sequences targeted by sgRNA are marked in green, while PAM sequences are marked in red; deleted nucleotides are indicated by hyphens, and dashed lines indicate areas that are omitted.
  • FIG. 10 DNA sequence analysis of mice obtained by editing the Y chromosome gene by the C-CRISPR method.
  • the tail DNA of the mouse obtained by targeting different genes on the Y chromosome was sequenced.
  • LED large fragment exon deletion
  • Indel insert or delete base
  • number on each column total number of sequenced TA clones.
  • Figure 11 Representative PCR products and sequences of monkey blastomeres, blastocysts and tissues obtained by targeting the gene Prrt2.
  • Embryos #4 obtained from sgRNA-Prrt2-C targeting and sgRNA-Prrt2-B+C+D targeted PCR products amplified from blastomeres of embryo #3.
  • the PCR products of the blastomeres were all TA cloned and sequenced.
  • sgRNA-Prrt2-B+C+D Targeted DNA sequences representative results for ears, tails and blood cells of monkeys #11 and #12. Sequences targeted by sgRNA are marked in green, while PAM sequences are marked in red; deleted nucleotides are indicated by hyphens, and dashed lines indicate areas that are omitted.
  • C Schematic representation of sgRNA targeting in COS-7 cells. After Cas9, sgRNA and mCherry were transferred into COS-7 cells, mCherry-positive cells were sorted and used for T7E1 analysis.
  • the arrows indicate the location of the T7E1 product action.
  • the control is normal COS-7 cell genomic DNA.
  • G Representative sequences of eight-cell embryos obtained by Arntl-X+X+X targeting. Sequences targeted by sgRNA are marked in green, while PAM sequences are marked in red; deleted nucleotides are indicated by hyphens, and dashed lines indicate areas that are omitted.
  • mice targeted by sgRNA-Tyr-B+C+D+E were used for off-target analysis.
  • the inventors selected up to 10 possible mutation sites for each sgRNA. PCR products were amplified from these genomic sites, TA cloned for ligation, and sequenced. Red: Not paired with the targeting sequence.
  • the inventors have intensively studied for the first time to disclose a cocktail-type CRISPR/Cas9 system and a gene knockout animal (without chimerism or low-integration) in which the gene function of the target gene is completely knocked out or most knocked out using the system.
  • the method of combining gene knockout animals ).
  • mammalian refers to a mammalian animal, including humans, non-human primates (monkeys, orangutans), livestock and farm animals (eg, pigs, sheep, cattle), rats ( Mice), as well as rodents (eg, mice, rats, rabbits).
  • Gene function completely knocks out the individual in the animal, and the function of each cell is destroyed, but the genotype of each cell is not necessarily identical.
  • a target gene refers to a gene of interest in an animal genome that requires a knockout operation.
  • target site on a target gene refers to a fragment in a “target gene”, and the sgRNA designed based on the “target site on the target gene” can recognize the target site, thereby Cleavage of the protein encoded by Cas9 occurs at this position.
  • the “target site on the target gene” is 18-26 nucleotides in length.
  • the "sgRNA” is either “Single-guide RNA (sgRNA)” or “single-directed RNA", which is based on a "target site on a target gene” design that contains sufficient sequence Synergistic with endonuclease Cas9 leads to the occurrence of Cas9-mediated DNA double-strand breaks at the target site.
  • sgRNA Single-guide RNA
  • single-directed RNA which is based on a "target site on a target gene” design that contains sufficient sequence Synergistic with endonuclease Cas9 leads to the occurrence of Cas9-mediated DNA double-strand breaks at the target site.
  • allele refers to a pair of genes occupying the same locus on a pair of homologous chromosomes that control a pair of relative traits. When a gene has two identical alleles, the individual is said to be homozygous for the gene or allele. When a gene of a body has two different alleles, the individual is said to be heterozygous for the gene.
  • the "low chimeric rate knockout animal” is an animal in which cells in which the target gene is to be knocked out without effective knockout account for less than 20% of the total number of cells; more preferably 15 % or less; more preferably 10% or less; further preferably 5% or less. More specifically, in the present invention, the "low chimerism gene knockout animal” is an animal obtained only after one birth cycle, that is, an animal of the F0 generation.
  • large fragment deletion refers to the presence of a contiguous base deletion greater than or equal to 30 bp in the gene of interest following performing a gene editing procedure as described herein.
  • insertion deletion refers to the presence of a contiguous base deletion of less than 30 bp in a gene of interest following performing a gene editing procedure as described herein.
  • C-CRISPR CRISPR/Cas9 system
  • C-CRISPR is based on CRISPR/Cas9 technology, by cocktails of Cas9 mRNA and two or more sgRNAs. The mixture is introduced into animal cells (especially animal fertilized egg cells) to obtain a method for efficient gene knockout efficiency.
  • the CRISPR/Cas9 system is a very efficient method of gene editing, but most of the genetically edited animals have chimerism, meaning that only a subset of their genes have been edited.
  • the present inventors have found that by introducing Cas9 mRNA into animal cells (especially fertilized eggs) and a plurality of contiguous (generally, 10-300 bp) separate targeting RNAs directed against key exons of each gene ( Single-guide RNA, sgRNA, can achieve knockout rates of up to 100% for one or more genes in animal embryos.
  • the present invention provides a method of preparing a knockout animal cell, the method comprising: (1) preparing two or more target genes on a target according to a nucleic acid sequence of a target gene to be knocked out a sgRNA at a site; and (2) co-transforming the sgRNA of (1) or a nucleic acid (eg, DNA) capable of forming the sgRNA, Cas9 mRNA, or a nucleic acid (eg, DNA) capable of forming the Cas9 mRNA into an animal cell In the case, the knockout animal cells are obtained.
  • a nucleic acid eg, DNA
  • the present invention also provides a method for preparing a gene knockout animal which completely knocks out or most knocks out a gene of a target gene, the method comprising: (1) preparing two or according to a nucleic acid sequence of a target gene to be knocked out a plurality of sgRNAs targeting different target sites on the target gene; (2) sgRNA of (1) or a nucleic acid (such as DNA) capable of forming the sgRNA, Cas9 mRNA or a nucleic acid capable of forming the Cas9 mRNA (such as DNA) Transferring into the fertilized egg, obtaining the fertilized egg of the knockout animal; (3) Generating the fertilized egg of (2) to produce the gene function of the target gene completely knocked out or most knocked out the knockout animal .
  • a nucleic acid such as DNA
  • the animal cell is an animal fertilized egg
  • the fertilized egg can be developed as a gene knockout animal whose target function is completely knocked out or most knocked out.
  • sgRNA target sites lead to higher gene editing efficiency, so it is important to design and find the right target site before proceeding with gene editing.
  • preparation of sgRNA is a technique known in the art, some software is currently available for the assisted design of sgRNA, but the selection of a suitable target site is still critical and difficult to achieve by software analysis alone. After designing a specific target site, in vitro cell viability screening is also required to obtain an effective target site for subsequent experiments.
  • the target sites on the target gene targeted by the sgRNA are between 9 and 500 bp apart. More suitable.
  • the interval may be 10 to 300 bp; for example, 15 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp, 150 bp, 180 bp, 200 bp, 250 bp, 280 bp. It should be understood that depending on the different situations of the gene, different lengths and different distributions of the exons, there may be other options, and those skilled in the art can obtain suitable choices according to the technical schemes given by the present invention.
  • the number of sgRNAs is generally between 3 and 30, for example, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25 articles.
  • the plurality of sgRNAs targeting different target sites on the target gene wherein at least 2, preferably at least 3, target sites on the target gene are located in the same explicit Sub-area.
  • the exon is a key exon of the target gene, which is edited to cause a significant change or loss of function of the gene function.
  • the plurality of sgRNAs targeting different target sites on the target gene are capable of introducing a frameshift mutation in a target site region of the target gene to which they are targeted.
  • designing the sgRNA within the functional or coding region of the gene of interest facilitates introduction of a frameshift mutation.
  • the plurality of sgRNAs targeting different target sites on the target gene are capable of introducing a large fragment deletion in a target site region of the target gene to which they are targeted.
  • the large fragment is deleted as a large fragment of the exon region.
  • the inventors have found that relatively short-distance contiguous sgRNAs can result in high-frequency exon large fragment deletions rather than common insertional deletions, which results in a higher probability of whole-gene deletion and more efficient gene knockout efficiency. .
  • the plurality of sgRNAs targeting different target sites on the target gene can simultaneously introduce an insertion deletion and a large fragment deletion in a target site region of the targeted target gene.
  • the C-CRISPR method of the invention preferably satisfies one or more of the following conditions: (a) using one exon of a plurality of sgRNA targeting genes; (b) multiple sgRNA targets Several sites closer to each other on the exon (interval as described above); (3) key exons of the targeted gene, sometimes only one or two amino acid mutations in the key domain of the protein It can completely disrupt the function of proteins; (4) pre-screening efficient sgRNAs by detecting the efficiency of sgRNA cleavage in embryos, especially for monkey genetic editing. These conditions can also result in the deletion of large exon fragments in addition to the general insertional deletion, which in turn allows the gene to be completely knocked out.
  • sgRNA and Cas9 can be employed to introduce sgRNA and Cas9 into the cell.
  • Some ways to introduce cells that can be considered are: transient expression, such as transfection, electroporation, or non-integrating viruses (AAV or adenovirus).
  • AAV or adenovirus non-integrating viruses
  • microinjection When applied to fertilized egg cells, it is preferred to use microinjection to introduce sgRNA and Cas9 into the cell.
  • the nucleic acid capable of forming the sgRNA is a nucleic acid construct or an expression vector
  • the nucleic acid capable of forming the Cas9 mRNA is a nucleic acid construct or an expression vector
  • the expression vector is introduced into the cell. Internally, active sgRNA and Cas9 mRNA are formed in the cells.
  • Cas9 mRNA carrying a promoter and sgRNA carrying a promoter can be obtained in vitro and injected into a cell.
  • promoters include, but are not limited to, the T7 promoter, the U6 promoter.
  • the method of the present invention can be used for preparing a knockout animal cell, an animal fertilized egg or an animal, wherein the animal is a knockout animal whose gene function is completely knocked out or most knocked out of the target gene;
  • the cells to be knocked out of the target gene are not effectively knocked out to account for 20% or less of the total number of cells; more preferably 15% or less; more preferably 10% or less; further preferably Ground below 5%.
  • the gene knockout of the target gene or the knockout of the most knockout animal is an animal obtained only after one birth cycle.
  • the gene function of the target gene obtained by the method of the present invention is completely knocked out or the most knocked out knockout animal can be used for carrying out animal gene function research, understanding various aspects of animal growth and development, and interested in various states.
  • the methods of the present invention can be used to perform animal embryo development studies to understand the function of genes of interest during embryonic development.
  • the method of the present invention can be applied to target a variety of genes of interest that require functional research, including but not limited to: reporter genes (such as fluorescent protein genes), phenotypic marker genes, structural genes, functional genes, etc. Knockout to obtain non-chimeric or chimeric knockout animals of the F0 generation.
  • the genes include, but are not limited to, GFP, Tyr, Tet1, Tet2, Tet3, Prrt2, ArntL, Y chromosome genes (eg, Zfy1, Ube1y1, Kdm5d, Eif2s3y, Ddx3y, Usp9y, Sry, Erdr1) ).
  • the CRISPR/Cas9 system is a very effective method of gene editing, but most of the genetically edited animals show chimerism, which prevents people from efficiently performing phenotypic analysis of gene knockouts.
  • animals injected with CRISPR/Cas9 can exhibit efficient gene deletion in nearly 100% of all cells, regardless of whether a single gene or multiple genes are deleted. This will be highly ethical, especially when using non-human primates as animal models.
  • phenotypic analysis of F0 mice knocking out eight genes on the Y chromosome (such as Zfy1, Ube1y1, Kdm5d, Eif2s3y, Ddx3y, Usp9y, Sry, Erdr1), respectively, fully demonstrates
  • the method of the invention is stable in the manufacture of knockout animals. Importantly, this method enables efficient knockout of the entire gene.
  • Phenotypic marker genes are, for example, fluorescence of GFP, skin whitening of Tyr, 5'-hydroxymethylation of cytosine of Tet-1, 2, 3, sex determination of Sry, and the like. The results of these examples all indicate that the target gene can be completely knocked out in embryos and animals, and complete loss of function can be achieved.
  • single-blast embryos or single-split genotypes of genetically engineered murine and monkey embryos are also analyzed, wherein all cells have biallelic mutations of the target gene, most of which (80 %) has large exon deletions, which in any case is strong evidence of complete gene deletion. Large exon deletions of exons can greatly increase the likelihood of loss of gene function compared to conventional insertional deletions, as this may result in a gene product that has no frameshift mutation but loss of function.
  • the Prt2 gene was knocked out by the C-CRISPR method, and a monkey model of human paroxysmal non-motor-induced dyskinesia (human motion disorder, PKD) was constructed without chimerism in one step.
  • PKD human paroxysmal non-motor-induced dyskinesia
  • PKD was found to be a single gene-induced neurological disease, rooted in a mutation in the Prrt2 gene. Building a monkey model of PKD will help to understand the pathological mechanisms of PKD and develop potential therapeutic approaches. To the best of the inventors' knowledge, this is the first time that CRISPR technology has been successfully used to obtain knockout monkeys with disease behavior phenotypes. The high efficiency of C-CRISPR demonstrated here makes genetic research among F0 generation monkeys no longer a dream.
  • the invention also provides a kit for preparing a gene knockout or a majority knockout gene knockout animal of a target gene, wherein the kit comprises a gene for interest, and is applied to perform C-
  • the CRISPR method operates sgRNA and Cas 9 mRNA or an agent capable of forming the sgRNA and Cas 9 mRNA in vivo or in vitro.
  • the genes of interest include: GFP, Tyr, Tet1, Tet2, Tet3, Prrt2, ArntL, Zfy1, Ube1y1, Kdm5d, Eif2s3y, Ddx3y, Usp9y, Sry, Erdr1 and the like.
  • the inventors have obtained in-depth research and screening, respectively obtained sgRNAs suitable for efficient knockout, and established a C-CRISPR system.
  • kits to facilitate use by those skilled in the art, such as microinjection reagents and the like.
  • instructions for use by those skilled in the art may also be included in the kit.
  • the Cas9 coding region on plasmid px260 was amplified by PCR using primers Cas9F and R (forward: TAATACGACTCACTATAGGGAGATTTCAGGTTGGACCGGTG; reverse: GACGTCAGCGTTCGAATTGC) while the T7 promoter was added to the product.
  • the T7-Cas9 PCR product was purified and used as a template for in vitro transcription (IVT) as mRNA, and the kit used was mMESSAGE mMACHINE T7ULTRA kit (Life Technologies) to obtain Cas9 mRNA.
  • Plasmid px330 (obtained from Addgene) was amplified by PCR using primers in the list (Table 1) while the T7 promoter was added to the sgRNA template.
  • the T7-sgRNA PCR product was purified and used as a template for in vitro transcription (IVT) sgRNA using the kit MEGA shortscript T7kit (Life Technologies) to obtain sgRNA.
  • the sgRNA designed for the corresponding gene When applied to an injection, the sgRNA designed for the corresponding gene, together with Cas9 mRNA, is microinjected into the fertilized egg.
  • mice When genetically editing mice, first hormones were superoved C57BL/6 female mice (three weeks old) or B6D2F1 (C57BL/6X DBA2J) female mice (7-8 weeks old), and then with C57BL/6 male mice or B6D2F1, respectively. The male rats mate and then collect the fertilized eggs from the fallopian tubes.
  • homozygous Actin-GFP transgenic male mice Okabe, M., et al. (1997). FEBS letters 407, 313-319) were mated with wild-type mother mice to collect fertilized eggs.
  • Cas9 mRNA (50 ⁇ g/ ⁇ l) and sgRNA (50 ⁇ g/ ⁇ l) were mixed and injected into the cytoplasm of the fertilized egg which can be clearly seen in the pronucleus by continuous flow pattern of FemtoJet microinjector (Eppendorf).
  • the injection process was carried out in cells containing 5 ⁇ g/ml.
  • the relaxin (CB) was completed in HEPES-CZB medium, and the injected embryos were raised to 1.5-day two-cell embryos in amino acid-containing KSOM medium at 37 ° C, 5% CO 2 . Subsequently, 25-30 two-cell embryos were transplanted into the fallopian tubes of a 0.5-day pseudopregnant ICR mother.
  • Ovarian collection was performed using a laparoscope when genetic editing of monkeys. After 32-36 hours of hCG stimulation, oocytes were aspirated from 2-8 mm diameter follicles and the collected oocytes were cultured in pre-equilibrated mature medium. Oocytes arrested in metaphase II of meiosis were used for intracytoplasmic sperm injection (ICSI), and then the appearance of two pronuclei was observed to determine whether they were fertilized. Cas9 mRNA (100 ⁇ g/ ⁇ l) and sgRNA (50 ⁇ g/ ⁇ l) were injected into the fertilized eggs. After the injection, the embryos were placed in HECM-9 medium and cultured until the next day. Some embryos are cultured to the mulberry or blastocyst stage, and then the extracted genome is collected and analyzed.
  • ICSI intracytoplasmic sperm injection
  • Single cells were collected and transferred using a glass tube under a stereo microscope.
  • the 8-16 cell stage embryo of the mouse or monkey was removed by bench acid digestion to remove the zona pellucida, then the embryo was transferred to 0.25% trypsin and gently pipetted to isolate a single blastomere.
  • the final blastomere is washed 7 to 10 times in 0.25% trypsin and transferred to a PCR tube.
  • Fibroblasts or leukocytes are gradually diluted with KSOM until the cells are completely dispersed. After 7 to 10 washes in KSOM, individual cells were transferred to a PCR tube.
  • lysate (0.1% Tween 20, 0.1% Triton X-100 and 4 ⁇ g/ml Proteinase K) was added to the PCR tube and then centrifuged to facilitate mixing. The mixture was reacted at 56 ° C for 30 minutes, followed by a reaction at 95 ° C for 5 minutes. The lysate was used as a template for the next nested PCR.
  • the genotype of the mutant mouse was detected by extracting the genomic DNA of the tail tissue and then performing a PCR reaction (primers for detection are shown in Table 3).
  • the ExTaq was activated at 95 ° C for 3 minutes, and the PCR procedure was 95 ° C for 30 s, 62 ° C for 30 s and 72 ° C for 1 minute, after repeated 34 cycles, and finally extended at 72 ° C for 5 minutes.
  • the PCR product was purified by tapping and DNA sequencing.
  • blastocysts After 6 washes of KSOM, individual blastocysts were transferred into a PCR tube containing 1.5 ⁇ l of lysate (0.1% Tween 20, 0.1% Triton X-100 and 4 ⁇ g/ml proteinase K) and then reacted at 56 ° C. Minutes, followed by a reaction at 95 ° C for 10 minutes to inactivate proteinase K. Nested primers were used for PCR amplification.
  • the ExTaq was activated at 95 ° C for 3 minutes, and the PCR procedure was 95 ° C for 30 s, 62 ° C for 30 s and 72 ° C for 1 minute, after repeated 34 cycles, and finally extended at 72 ° C for 5 minutes.
  • the second round of PCR reaction was carried out using the previous round of 0.5 ⁇ l PCR product as a template and the nested internal primer as a PCR primer.
  • the PCR reaction system is the same as before.
  • testes were fixed overnight with bouin's solution.
  • the fixed tissue was embedded in paraffin and then cut into a thickness of 5 ⁇ m using a Microtome. After dewaxing, the rehydrated sections were stained with hematoxylin-eosin.
  • 5hmC staining the embryos were excised from the pregnant female rats at a specific number of days, then fixed with 4% paraformaldehyde, and embedded with OCT (Sakura), and cut into a thickness of 8 ⁇ m.
  • the sections were treated with hydrochloric acid solution (4N hydrochloric acid, 0.1% Triton X-100 in distilled water), followed by washing with PBS, and blocking solution (1% BSA, 10% goat serum and 0.3% Triton X-100). PBS) is transparent.
  • the sections were incubated with primary antibody (mouse anti-5 mC antibody, 1:500, Eurogentec #BI-MECY-0100; rabbit anti-5hmC antibody, 1:1000, Active Motif #39792) at 4 ° C overnight, and the secondary antibody was incubated at room temperature. One hour.
  • these sections were mounted with an anti-quenching agent (Invitrogen) and photographed with a LEICA TCS SP5 II confocal microscope. The intensity of the signal was determined using the Leica Application Suite software.
  • the sperm was diluted to 3-6 x 10 6 /ml with this buffer and then incubated for 20 minutes in a dish at 37 °C.
  • the sperm suspension is gently mixed before measuring the viability.
  • For each vitality measurement use a large-aperture tip to take a drop of 5 ⁇ l of sperm suspension into the card slot of a pre-heated Lejia slide (depth, 10 ⁇ m), and then use a computer.
  • Auxiliary Semen Analysis (CASA) instrument HTM-TOX IVOS sperm viability analyzer, Animal Motility, version 12.3A; Hamilton Thorne Research). The magnification is 10 ⁇ . All samples are counted at least twice to avoid errors due to incorrect sampling. Record at least five areas and at least 100 viable spermatozoa per sample. The image is saved and analyzed for subsequent analysis.
  • Prrt2 mutant monkey The founders of the Prrt2 mutant monkey (F0) began to observe behavior since their birth. Typical anomalous features are recorded with a camera (Sony HDR-XR520 Handicam). Each monkey was placed in a platform or observation box (1.5x 1x 1.1m) for recording according to different observation purposes.
  • the primary antibodies used here were: rabbit anti-PRRT2 antibody (1:2000, Sigma) and HRP-conjugated anti-GAPDH antibody (1:8000, Kangchen).
  • rabbit anti-PRRT2 antibody (1:2000, Sigma)
  • HRP-conjugated anti-GAPDH antibody (1:8000, Kangchen).
  • each of the membranes was washed 3 times with TBST for 10 minutes each time, and then HRP-conjugated anti-rabbit secondary antibody was used for two hours at room temperature. After washing again, the strips can be developed using ECL Plus Western Blot detection reagent (Tiangen).
  • Cos-7 cells were seeded into 6-well plates 24 hours prior to transfection.
  • Cells were transfected with plasmids at 80-90% with Lipofectamine 3000 (Life Technologies).
  • Cells were transiently transfected with the pX330-mCherry-sgRNA plasmid (established based on addgene 42230).
  • 72 hours after transfection mCherry-positive cells were sorted by flow cytometry, and the cells were resuspended in lysate (0.1% Tween 20, 0.1% Triton X-100, and 4 ⁇ g/ml proteinase K) and reacted at 56 °C. Minutes, followed by a reaction at 95 ° C for 10 minutes to inactivate proteinase K.
  • the SURVEYOR method is consistent with the previously reported steps.
  • the primers used for PCR amplification are detailed in Table 3.
  • the inventors first wanted to know whether pre-injection of Cas9 mRNA and sgRNA into the mouse MII egg in the fertilized egg could reduce the chimerism.
  • the targeted genes are Tet1, Tet2; the primers used to make the in vivo transcription template are the corresponding sequences in Table 1 (Tet1 and Tet2 and sgRNA-R); the sgRNA target sequences are the corresponding sequences in Table 2; the primers applied to genotype analysis The corresponding sequences are shown in Table 3.
  • Two-step injection Cas9 mRNA and sgRNA were injected into mouse MII oocytes, and sperm were injected into MII oocytes 4 hours later.
  • One-step injection injection of Cas9 mRNA and sgRNA into mouse zygotic embryos. As a result, no significant effect was observed, and the chimerism was not significantly lowered, as shown in Figs. 7A-F.
  • the inventors adjusted the design of various sgRNA experiments, the effect was still not satisfactory. In turn, the inventors tried to use multiple sgRNAs instead of one sgRNA to see if it could reduce the chimerism of CRISPR/Cas9 editing embryonic genes. In order to be able to easily estimate the chimeric rate, the present inventors used a homozygous Actin-EGFP male transgenic mouse (widely expressing GFP throughout the body) and a wild-type female mouse to obtain a fertilized egg, and simultaneously injected Cas9 mRNA and GFP-targeting sgRNA was analyzed for GFP expression at the blastocyst stage (Fig. 1A).
  • the inventors designed four sgRNAs directed against the same exon of GFP, the target sequences of which are 10-200 bp apart from each other.
  • the primers used to make the in vivo transcription template are the corresponding sequences in Table 1 (GFP-A, GFP-B, GFP-C, GFP-D); the sgRNA target sequences are the corresponding sequences in Table 2 (GFP-A, GFP-B, GFP-C, GFP-D); primers for genotypic analysis are the corresponding sequences (GFP) in Table 3.
  • the inventors targeted the Tyrosinase gene (Tyr for pigmentation) of the fertilized egg, and obtained the gene editing by two-cell embryo transfer. Mice (Fig. 2A). Mice with one or two copies of wild-type Tyr showed complete coloration. Conversely, mice with all two alleles failed were whitened, so the chimerism of the mice was visually observed.
  • the primers used to make the in vivo transcription template are the corresponding sequences in Table 1 (Tyr-A ⁇ Tyr-F and sgRNA-R); the sgRNA target sequences are the corresponding sequences in Table 2.
  • sgRNA target sites A (Tyr-A) and F (Tyr-F) are located next to the intron of the exon 4; sgRNA target site B (Tyr-B), C (Tyr- C), D (Tyr-D), and E (Tyr-E) are located in exon 4.
  • the inventors also compared the effect of increasing sgRNA on the birth rate of mice, and found that increasing the sgRNA of the targeted gene did not reduce the birth rate of the mouse (30-50%, Figure 2E).
  • mice After mating with ICR mice (albino lines), the genetically modified mice targeted by four sgRNAs (sgRNA-Tyr-B+C+D+E) have normal fertility and 100% of the offspring are albino, indicating that these mice The Tyr gene in the germ cells was completely knocked out (Table 4).
  • C-CRISPR can efficiently produce knockout mice without chimerism in the F0 generation.
  • the experimental data of the above five groups indicate that the introduction of frameshift mutations and large exon deletions can lead to efficient genes. delete.
  • the inventors performed DNA sequence analysis on embryos and mice edited by Tyr gene. After identifying the tail tissues of 6 four-sgRNA-targeted Tyr mice (sgRNA-Tyr-B+C+D+E-targeted albino mice), the inventors found that Tyr in five mice Knockout is entirely due to knockout of the entire exon, while 80% of Tyr knockout in the remaining mouse is due to exon knockout and 20% is due to insertional deletion. (Figs. 3A and 3B; Figs. 8A and 8B).
  • the inventors further detected genetically engineered embryos at the single cell level.
  • the inventors determined the Tyr gene of each blastomere of 8 to 16 cell stage embryos by PCR, and approximately 53% of blastomeres were successfully amplified (Fig. 8G and Fig. 8H).
  • Detection of blastomeres from 21 embryos from 3 to 4 sgRNA targeting groups (Group IV) revealed that each blastomere (n 116) exhibited a deletion of the wild-type Tyr gene, both inserted Deletion and deletion of large exon fragments, or both ( Figures 3E to 3I).
  • Tet1-A ⁇ C The primers used to make the in vivo transcription template are the corresponding sequences in Table 1 (Tet1-A ⁇ C; Tet2-A-C; Tet3-A-C; and sgRNA-R); the sgRNA target sequences are the corresponding sequences in Table 2.
  • the inventors measured the average ratio of 5hmC/5mC using the immunofluorescence density, and the results showed that the ratio was halved in the groups I and II, and became lower in the group III (Fig. 4D).
  • the inventors then used the C-CRISPR method to perform phenotypic analysis of the functions of a plurality of Y chromosome genes, and also tested the efficiency of one-step gene knockout in mice for rapid functional screening of a large number of genes.
  • the Y chromosome is highly specialized and is dedicated to male sex differentiation and fertility. However, to date only a few genes have been targeted for knockout and tested for their biological effects. Therefore, the inventors decided to target eight single copy Y chromosome genes therein: Zfy1; Ube1y1; Kdm5d; Eif2s3y; Ddx3y; Usp9y; Sry; Erdr1 (Fig. 5A).
  • the primers used to make the in vivo transcription template are the corresponding sequences in Table 1 (Zfy1-A ⁇ B; Ube1y1-A ⁇ B; Kdm5d-A ⁇ B; Eif2s3y-A ⁇ B; Ddx3y-A ⁇ B; Usp9y-A ⁇ B ; Sry-A ⁇ B; Erdr1-A ⁇ B; and sgRNA-R); sgRNA target sequences are the corresponding sequences in Table 2.
  • mice targeting the other six genes had a normal sex ratio ( Figures 5B and 5C). Male mice targeted for deletion of the Eif2s3y gene were infertile and testicular dysplasia (Fig.
  • mice that deleted the other five genes were fertile and could be mated with wild-type female mice to produce offspring (Table 5, Table 6).
  • mice that deleted the Zfy1 or Kdm5d gene had lower testicular body weight, whereas mice that deleted the Ube1y1 or Dxd3y gene had lower viability of normal sperm (Fig. 5D to 5F, Table 5).
  • Histological analysis of testicular tissue also indicated that only the Eif2s3y gene of the six genes caused defects in spermatogonial differentiation (Fig. 5G).
  • Example 6 One-step method to obtain a chimeric monkey with no chimerism
  • the present inventors examined whether the C-CRISPR method can be used to construct non-chimeric gene-editing monkeys.
  • the present inventors made a targeted knockout of the Prrt2 gene of cynomolgus monkey (Macaca fascicularis) in order to construct an animal model of Paroxysmal Kinesigenic Dyskinesia (PKD).
  • the inventors first designed ten sgRNAs (referring to one sgRNA per injection) for the exon 2 of the Prrt2 gene using the traditional CRISPR/Cas9 strategy, and injected the embryos (one for each injection) into these sgRNAs, and then Detection is performed at the blastocyst stage.
  • the primers used to make the in vivo transcription template are the corresponding sequences in Table 1 (Prrt2-A and sgRNA-R); the sgRNA target sequences are the corresponding sequences in Table 2.
  • sgRNA-Prrt2-A was highly efficient in targeting genes (Fig. 6A and Fig. 11).
  • the inventors injected sgRNA-Prrt2-A and Cas9 mRNA into monkey fertilized eggs together, and transplanted 55 such embryos into 17 surrogate mothers in the two-cell stage. Received 6 abortions and 4 live monkeys. After genomic analysis of the tails of ten monkeys, it was found that two stillbirths (#2 and #3) and three live monkeys (#4, #8, and #10) had 20-60% different degrees of Prrt2 deletion. The remaining 5 monkeys were not genetically edited (Fig. 6F, Table 7).
  • the inventors In order to try the C-CRISPR method, the inventors additionally constructed three sgRNAs (sgPrrt2-B to sgPrrt2-D) targeting the exon 3 of the Prrt2 gene, and injecting these sgRNAs in the fertilized eggs, etc. The efficiency is detected by the blastocyst stage.
  • the primers used to make the in vivo transcription template are the corresponding sequences in Table 1 (Prrt2-B to D); the sgRNA target sequences are the corresponding sequences in Table 2.
  • the inventors have found that all of these sgRNAs are capable of introducing DNA cleavage at the targeting site (Fig. 11A).
  • control group that is, the blastomeres of eight eight-cell embryos injected with only a single sgRNA (sgPrrt2-C), were 90% wild-type, and another 10% were single allele mutations, with high chimerism.
  • the characteristics of sex were consistent (3 chimeric embryos and 5 wild-type embryos) (Fig. 6B to 6E; Table 7).
  • a homozygous mutation in the PRRT2 gene in a patient is accompanied by a series of clinical symptoms such as various paroxysmal dyskinesia, benign familial infantile epilepsy, hemiplegia migraine, paroxysmal torticollis, paroxysmal ataxia Even mental retardation occurs.
  • Prrt2 knockout monkey was examined whether the Prrt2 knockout monkey can also exhibit these symptoms.
  • the present inventors observed an abnormality in motor function similar to paroxysmal dyskinesia: survival of monkey #11 by sgRNA-Prrt2-B+C+D was not apparently defective, and was born shortly after birth. During a period of time, it showed normal activity (Fig. 6H).
  • the monkey After 18 days, the monkey showed symptoms similar to paroxysmal exercise-induced dyskinesia, with abnormal movements of the limbs (torso bending and occasional tight-fitting fists). This action is not observed in wild-type monkeys of the same age, and these abnormal movements may be induced by sudden spontaneous movements (similar to PKD symptoms).
  • Monkey #11 At 61 days of age, Monkey #11 showed severe dyskinesia, including limb tremors and lower limb stiffness that prevented the monkey from walking and sitting down. At 105 days, Monkey #11 resumed walking ability after receiving lower limb training.
  • PKD-like symptoms disappeared, but under certain stresses, such as flipping or swinging tails, they can also cause abnormal limb movements and Paroxysmal nonkinesigenic dyskinesia (PNKD).
  • PNKD Paroxysmal nonkinesigenic dyskinesia
  • the inventors have also observed abnormal behaviors such as hand and foot clenching fists during touch and extreme sleepiness.
  • Monkey #11 developed a headache-like symptom that often hits the contents of the cage or the cage floor with his head, while clenching his fist and shaking his body. This headache-like symptom lasts for about a month. .
  • Monkey #11 also had a learning disability at the age of one, because it could not learn to use hands and eat food, so it was eaten by lowering the head and getting close to the food on the floor instead of using it. Many of these phenotypes are similar to those that exhibit abnormalities associated with human Prrt2.
  • #12 monkeys obtained by injecting a single sgRNA with the CRISPR/Cas9 system and #12 monkeys obtained by the C-CRISPR method were the same as chimeras, and during the observation period of up to 1 year, No dyskinesia was found.
  • the inventors In order to demonstrate the reliability of complete knockout in monkey embryos, the inventors also used the C-CRISPR method to target the aromatic hydrocarbon receptor nuclear transporter-like protein 1 (Arntl), which is a core component of the rhythm clock.
  • the inventors first designed four sgRNAs (sgRNA-Arntl-A, B, G and J) to target two functional PAS domains of Arntl (exons 8 and 13 respectively) and then in monkeys DNA shear efficiency was measured in the embryo (Fig. 12A).
  • sgRNA-Arntl-G and sgRNA-Arntl-J acted significantly on exon 13 (Fig. 12B).
  • the inventors designed an additional 7 sgRNAs (sgRNA-Arntl-C, D, E, F, H, I and K) for exon 13 (Fig. 12A).
  • the sgRNA cleavage efficiency was detected in the monkey cell COS-7 by the T7E1 assay, and the inventors found that, like the two sgRNAs (sgRNA-Arntl-G and J) previously tested on monkey embryos, five sgRNAs (sgRNA) -Arntl-D, E, H, I and K) have high DNA shear efficiency (37% to 60%) ( Figures 12C and 12D). Considering the distance between each sgRNA, the inventors selected sgRNA-Arntl-D, sgRNA-Arntl-G, and sgRNA-Arntl-J to co-inject into monkey embryos, and then perform genes in embryos of eight-cell stage.
  • Prrt2 contains large fragment exon deletions in approximately 50-80% of cells, with the remainder showing only Prrt2 point mutations (335N ⁇ 335A).
  • the inventors also performed whole genome sequencing (WGS) on the samples, and no off-target effects were found at the 20 sites most likely to be off target.
  • GGS whole genome sequencing

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Environmental Sciences (AREA)
  • Mycology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un système CRISPR/Cas9 (C-CRISPR) de type cocktail et un procédé de préparation d'un animal à invalidation génique, dont la fonction génique est complètement inactivée ou quasiment inactivée, à l'aide du système.
PCT/CN2018/081016 2017-03-31 2018-03-29 Procédé de préparation d'un animal à invalidation génique non chimérique basé sur la technologie crispr/cas9 Ceased WO2018177351A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710207362.1A CN108660161B (zh) 2017-03-31 2017-03-31 基于CRISPR/Cas9技术的制备无嵌合基因敲除动物的方法
CN201710207362.1 2017-03-31

Publications (1)

Publication Number Publication Date
WO2018177351A1 true WO2018177351A1 (fr) 2018-10-04

Family

ID=63675255

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/081016 Ceased WO2018177351A1 (fr) 2017-03-31 2018-03-29 Procédé de préparation d'un animal à invalidation génique non chimérique basé sur la technologie crispr/cas9

Country Status (2)

Country Link
CN (1) CN108660161B (fr)
WO (1) WO2018177351A1 (fr)

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
CN110862984A (zh) * 2019-11-05 2020-03-06 桂林医学院 一种特异靶向小鼠Tyr基因的sgRNA导向序列及其应用
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
CN116798511A (zh) * 2023-04-13 2023-09-22 西安交通大学 错配sgRNA选取方法及其在DNA特定位点上CRISPR系统中的应用
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12006520B2 (en) 2011-07-22 2024-06-11 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US12157760B2 (en) 2018-05-23 2024-12-03 The Broad Institute, Inc. Base editors and uses thereof
US12281338B2 (en) 2018-10-29 2025-04-22 The Broad Institute, Inc. Nucleobase editors comprising GeoCas9 and uses thereof
US12351837B2 (en) 2019-01-23 2025-07-08 The Broad Institute, Inc. Supernegatively charged proteins and uses thereof
US12390514B2 (en) 2017-03-09 2025-08-19 President And Fellows Of Harvard College Cancer vaccine
US12406749B2 (en) 2017-12-15 2025-09-02 The Broad Institute, Inc. Systems and methods for predicting repair outcomes in genetic engineering
US12435330B2 (en) 2019-10-10 2025-10-07 The Broad Institute, Inc. Methods and compositions for prime editing RNA
US12473543B2 (en) 2019-04-17 2025-11-18 The Broad Institute, Inc. Adenine base editors with reduced off-target effects

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109402244B (zh) * 2018-12-20 2022-05-03 广西大学 一种哺乳动物胚胎性别鉴定方法
CN111793654B (zh) * 2019-04-08 2022-05-24 中国农业大学 一种提高CRISPR/Cas9介导的双等位基因突变效率的方法及其应用
CN110423785A (zh) * 2019-05-29 2019-11-08 扬州大学 一种基于CRISPR-Cas9编辑技术的敲除鸡KPNA3基因的细胞系及其构建方法
CN111304172A (zh) * 2020-02-18 2020-06-19 扬州大学 一种基于CRISPR-Cas9编辑技术的敲除鸡EphA2基因的细胞系的构建方法
CN111235154A (zh) * 2020-04-01 2020-06-05 陕西慧康生物科技有限责任公司 靶向敲除人MC1R基因的sgRNA及其构建的细胞株
CN111235153B (zh) * 2020-04-01 2023-05-26 陕西慧康生物科技有限责任公司 靶向敲除人MC1R基因的sgRNA及其构建的细胞株
CN116064541B (zh) * 2022-10-21 2025-03-18 华南农业大学 一种基于CRISPR/Cas9系统敲除猪细胞Y染色体的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105400810A (zh) * 2015-09-06 2016-03-16 吉林大学 采用敲除技术建立低磷性佝偻病模型的方法
WO2016133165A1 (fr) * 2015-02-19 2016-08-25 国立大学法人徳島大学 PROCÉDÉ POUR TRANSFÉRER DE L'ARNm DE Cas9 DANS UN OVULE FÉCONDÉ DE MAMMIFÈRE PAR ÉLECTROPORATION

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0318688D0 (en) * 2003-08-08 2003-09-10 Chiron Srl Streptococcus pneumoniae knockout mutants
JPWO2008102602A1 (ja) * 2007-02-22 2010-05-27 国立大学法人 東京大学 Ｂｌａｓｔｏｃｙｓｔｃｏｍｐｌｅｍｅｎｔａｔｉｏｎを利用した臓器再生法
WO2010129670A2 (fr) * 2009-05-07 2010-11-11 Children's Medical Center Corporation Procédé de modulation de l'angiogenèse à l'aide de fibromoduline
CN113563476A (zh) * 2013-03-15 2021-10-29 通用医疗公司 遗传和表观遗传调节蛋白至特定基因组基因座的rna引导的靶向
US9663782B2 (en) * 2013-07-19 2017-05-30 Larix Bioscience Llc Methods and compositions for producing double allele knock outs
CN105039339B (zh) * 2015-06-05 2017-12-19 新疆畜牧科学院生物技术研究所 一种以RNA介导的特异性敲除绵羊FecB基因的方法及其专用sgRNA
CN105112446A (zh) * 2015-06-25 2015-12-02 中国医学科学院基础医学研究所 使用单倍体干细胞高效建立遗传修饰动物模型的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016133165A1 (fr) * 2015-02-19 2016-08-25 国立大学法人徳島大学 PROCÉDÉ POUR TRANSFÉRER DE L'ARNm DE Cas9 DANS UN OVULE FÉCONDÉ DE MAMMIFÈRE PAR ÉLECTROPORATION
CN105400810A (zh) * 2015-09-06 2016-03-16 吉林大学 采用敲除技术建立低磷性佝偻病模型的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CONG, L ET AL.: "Multiplex Genome Engineering Using CRISPR/Cas Systems", SCIENCE, vol. 339, no. 6121, 15 February 2013 (2013-02-15), pages 819 - 823, XP055067741, ISSN: 0036-8075 *
LIU, ZHIGUO: "Research Progress on CRISPR/Cas9 Mediated Genome Editing", CHINESE JOURNAL OF ANIMAL AND VETERINARY SCIENCES, vol. 45, no. 10, 15 October 2014 (2014-10-15), pages 1567 - 1581, ISSN: 0366-6964 *

Cited By (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12006520B2 (en) 2011-07-22 2024-06-11 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US12473573B2 (en) 2013-09-06 2025-11-18 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US12215365B2 (en) 2013-12-12 2025-02-04 President And Fellows Of Harvard College Cas variants for gene editing
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US12398406B2 (en) 2014-07-30 2025-08-26 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US12344869B2 (en) 2015-10-23 2025-07-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US12043852B2 (en) 2015-10-23 2024-07-23 President And Fellows Of Harvard College Evolved Cas9 proteins for gene editing
US11999947B2 (en) 2016-08-03 2024-06-04 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US12084663B2 (en) 2016-08-24 2024-09-10 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US12390514B2 (en) 2017-03-09 2025-08-19 President And Fellows Of Harvard College Cancer vaccine
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US12435331B2 (en) 2017-03-10 2025-10-07 President And Fellows Of Harvard College Cytosine to guanine base editor
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US12359218B2 (en) 2017-07-28 2025-07-15 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US12406749B2 (en) 2017-12-15 2025-09-02 The Broad Institute, Inc. Systems and methods for predicting repair outcomes in genetic engineering
US12157760B2 (en) 2018-05-23 2024-12-03 The Broad Institute, Inc. Base editors and uses thereof
US12281338B2 (en) 2018-10-29 2025-04-22 The Broad Institute, Inc. Nucleobase editors comprising GeoCas9 and uses thereof
US12351837B2 (en) 2019-01-23 2025-07-08 The Broad Institute, Inc. Supernegatively charged proteins and uses thereof
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US12281303B2 (en) 2019-03-19 2025-04-22 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US12473543B2 (en) 2019-04-17 2025-11-18 The Broad Institute, Inc. Adenine base editors with reduced off-target effects
US12435330B2 (en) 2019-10-10 2025-10-07 The Broad Institute, Inc. Methods and compositions for prime editing RNA
CN110862984B (zh) * 2019-11-05 2021-04-06 桂林医学院 一种特异靶向小鼠Tyr基因的sgRNA导向序列及其应用
CN110862984A (zh) * 2019-11-05 2020-03-06 桂林医学院 一种特异靶向小鼠Tyr基因的sgRNA导向序列及其应用
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12031126B2 (en) 2020-05-08 2024-07-09 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
CN116798511A (zh) * 2023-04-13 2023-09-22 西安交通大学 错配sgRNA选取方法及其在DNA特定位点上CRISPR系统中的应用

Also Published As

Publication number Publication date
CN108660161B (zh) 2023-05-09
CN108660161A (zh) 2018-10-16

Similar Documents

Publication Publication Date Title
WO2018177351A1 (fr) Procédé de préparation d'un animal à invalidation génique non chimérique basé sur la technologie crispr/cas9
CN110551759B (zh) 一种提高转基因细胞重组效率的组合物及方法
CN109628454B (zh) 斑马鱼糖原贮积症gys1和gys2基因突变体的构建方法
CN106929533A (zh) Kars基因点突变小鼠模型的构建方法及其应用
Aida et al. The dawn of non-human primate models for neurodevelopmental disorders
CN116103342A (zh) 基于CRISPR-Cas9系统和PB转座子系统的羊早期胚胎发育的谱系示踪方法
CN117025686A (zh) 一种胰岛β细胞Pik3r3基因条件性敲除小鼠模型的构建方法和应用
CN115807037A (zh) 一种遗传可控的四倍体鱼的选育方法及三倍体鱼的制备方法
CN117701634A (zh) 一种itgb5条件性基因敲除小鼠模型、构建方法及其应用
CN111979241A (zh) 制备视网膜色素变性非人哺乳动物模型的方法
CN114908098A (zh) 斑马鱼hoxb1a基因缺失突变体的制备方法和应用
CN115261360A (zh) 一种gata6基因敲除斑马鱼模型的构建方法
CN114480497B (zh) 一种ep400基因敲除斑马鱼心力衰竭模型的构建及其应用的方法
RU2764650C1 (ru) Способ получения линии гуманизированных мышей, содержащих инсерцию 3974insT в гене mGrin2a (mice glutamate [NMDA] receptor subunit epsilon-1), приводящую к преждевременному прекращению трансляции белка grin2a
CN110283851A (zh) 与恶性胸腔积液相关的靶点myo9b及其应用
US11246299B2 (en) Disease model pig exhibiting stable phenotype, and production method thereof
CN118813623B (zh) 一种ttc21b基因缺失动物模型的构建方法及其应用
Brakebusch Generation and analysis of genetically modified mice
US11732273B2 (en) Methods and compositions for in situ germline genome engineering
CN116376976A (zh) 人源IL32γ条件敲入小鼠模型的构建方法及其应用
CN118773200B (zh) 一种ttc21b基因突变位点及其应用
WO2021190226A1 (fr) Application d'une réparation d'épissage médiée par édition mono-base dans la préparation et le traitement de l'amyotrophie spinale
CN119769472B (zh) 一种Prpf6基因突变引发单倍剂量不足的RP小鼠模型的构建方法及其应用
CN111518839B (zh) 一种等位特异性位点编辑方法
CN111500580B (zh) 一种基因编辑方法

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: 18776261

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18776261

Country of ref document: EP

Kind code of ref document: A1