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WO2018177351A1 - 基于CRISPR/Cas9技术的制备无嵌合基因敲除动物的方法 - Google Patents

基于CRISPR/Cas9技术的制备无嵌合基因敲除动物的方法 Download PDF

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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
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sgrna
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gene
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target gene
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杨辉
熊志奇
孙强
左二伟
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Shanghai Institutes for Biological Sciences SIBS of CAS
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • 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
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    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
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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

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Abstract

提供了一种鸡尾酒式CRISPR/Cas9系统(C-CRISPR)及利用该系统制备基因功能完全敲除或大部分敲除的基因敲除动物的方法。

Description

基于CRISPR/Cas9技术的制备无嵌合基因敲除动物的方法 技术领域
本发明属于分子生物学领域，更具体地，本发明涉及基于CRISPR/Cas9技术的、于改造的第一代高效获得目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的方法。
背景技术
CRISPR/Cas9源自细菌和古细菌的免疫系统，可利用靶位点特异性的RNA将Cas9核酸酶带到基因组上的特定位置，从而实现对基因位点的精确切割。CRISPR/Cas9技术已被应用于疾病模型建立、药物靶点筛选、并正在成为新一代基因治疗手段。
CRISPR/Cas9系统已在多个物种中用于基因组编辑，目前人们已可以将Cas9 mRNA和sgRNA(single-guide RNA)直接注入原核期的受精卵从而在特定位置引入双链断裂(double-strand break，DSB)。CRISPR/Cas9引入的双链断裂会被一种叫做“非同源末端连接”(non-homologous end joining，NHEJ)的趋错修复机制所处理，致使基因突变的动物携带移码突变。然而，大部分用此方法得到的突变动物都有嵌合现象，也就是说他们的基因突变只存在于部分细胞而别的细胞则不然，这主要是因为所注的内切酶在卵裂之后才完成DNA的切割。对于涉及表型的研究而言，嵌合的基因编辑动物需要进一步地杂交育种，才能得到完全的基因敲除动物，当需要敲除多个基因的时候，这种操作就会变得颇为艰巨。当涉及大型动物，比方说非人灵长类的时候，嵌合体的问题就会显得尤为严重，因为猕猴的繁殖周期长达5-6年而且每胎只生一个幼崽，严重阻碍了针对这类动物的研究进程。
之前有许多研究者都试图通过一步法来产生没有嵌合的基因修饰动物，尤其是大型动物。包括将Cas9 mRNA和sgRNA注射到卵母细胞中而非受精卵中，或注射Cas9蛋白。但是DNA测序分析了这些方法的动物个体组织后，发现这些方法完全做到双等位基因敲除的效率相当低。
因此，本领域迫切需要寻找新的，更高效率的基因敲除方法，以期在更短的时间周期内获得基因功能完全敲除或大部分敲除的基因敲除动物。
发明内容
本发明的目的在于提供一种基于CRISPR/Cas9技术的、于改造的第一代高效获得基因功能完全敲除的基因敲除动物的方法。
在本发明的第一方面，提供制备基因敲除的动物细胞的方法，所述方法包括：(1)根据待敲除的目标基因的核酸序列，制备两条或多条靶向目标基因上不同靶位点的sgRNA；(2)将(1)的sgRNA或能形成所述sgRNA的核酸、Cas9 mRNA或能形成所述 Cas9 mRNA的核酸共转入动物细胞中，获得基因敲除的动物细胞。
在一个优选例中，所述的动物细胞为动物受精卵，所述的受精卵可发育为目标基因的基因功能完全敲除或大部分敲除的基因敲除动物。
在本发明的另一方面，提供制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的方法，所述方法包括：(1)根据待敲除的目标基因的核酸序列，制备两条或多条靶向目标基因上不同靶位点的sgRNA；(2)将(1)的sgRNA或能形成所述sgRNA的核酸、Cas9 mRNA或能形成所述Cas9 mRNA的核酸共转入受精卵中，获得基因敲除的动物受精卵；(3)使(2)的受精卵发育产生所述的目标基因的基因功能完全敲除或大部分敲除的基因敲除动物。
在一个优选例中，所述的两条或多条靶向目标基因上不同靶位点的sgRNA，靶向的目标基因上的靶位点之间间隔为9～500bp；较佳地为10～300bp，例如，15bp，20bp，30bp，40bp，50bp，60bp，70bp，80bp，90bp，100bp，120bp，150bp，180bp，200bp，250bp，280bp。
在另一优选例中，采用多条靶向目标基因上不同靶位点的sgRNA；如为3～30条，例如，4，5，6，7，8，9，10，12，15，18，20，25条。
在另一优选例中，所述的多条靶向目标基因上不同靶位点的sgRNA，其中至少2条(较佳地至少3条，例如，4，5，6，7，8，9，10，12，15，18，20，25条)所靶向的目标基因上的靶位点位于同一外显子区域。
在另一优选例中，所述的多条靶向目标基因上不同靶位点的sgRNA，在所靶向的目标基因上的靶位点区域引入移码突变和/或引入插入缺失和/或引入大片段缺失。
在另一优选例中，所述的能形成所述sgRNA的核酸为核酸构建体或表达载体，或所述的能形成所述Cas9 mRNA的核酸为核酸构建体或表达载体。
在另一优选例中，所述的动物为哺乳动物，包括(但不限于)：人，非人灵长类动物，鼠，家畜。
在另一优选例中，所述的sgRNA的核酸序列的上游携带启动子，较佳地该启动子为T7启动子，U6启动子；或所述的Cas9 mRNA的核酸序列的上游携带启动子，较佳地该启动子为T7启动子。
在另一优选例中，所述的目标基因的基因功能大部分敲除的基因敲除动物的体内，待敲除目标基因未发生有效敲除的细胞占总细胞数的20％以下；更佳的15％以下；更佳地10％以下；进一步更佳地5％以下。
在另一优选例中，所述的待敲除的目标基因为GFP基因，所述的sgRNA为2，3或4条，各sgRNA靶向的目标基因上的靶位点之间间隔为30～200bp；较佳地40～150bp；更佳地50～130bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的2，3或4个：SEQ ID NO:48，SEQ ID NO:49，SEQ ID NO:50，SEQ ID NO: 51。
在另一优选例中，所述的待敲除的目标基因为Tyr基因，所述的sgRNA为3，4，5或6条，各sgRNA靶向的目标基因的外显子4及任选地与该外显子邻近的内含子，且sgRNA靶向的靶位点之间间隔为10～100bp；较佳地10～70bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的3，4，5或6个：SEQ ID NO:52，SEQ ID NO:53，SEQ ID NO:54，SEQ ID NO:55，SEQ ID NO:56，SEQ ID NO:57；更佳地为选自下组的3或4个：SEQ ID NO:53，SEQ ID NO:54，SEQ ID NO:55，SEQ ID NO:56。
在另一优选例中，所述的待敲除的目标基因为Tet1基因，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子2，且各sgRNA靶向的靶位点之间间隔为80～200bp；较佳地100～190bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:58，SEQ ID NO:59，SEQ ID NO:60。
在另一优选例中，所述的待敲除的目标基因为Tet2基因，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子3，且各sgRNA靶向的靶位点之间间隔为100～150bp；较佳地110～135bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:61，SEQ ID NO:62，SEQ ID NO:63。
在另一优选例中，所述的待敲除的目标基因为Tet3基因，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子4，且各sgRNA靶向的靶位点之间间隔为180～280bp；较佳地190～270bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:64，SEQ ID NO:65，SEQ ID NO:66。
在另一优选例中，所述的待敲除的目标基因为Prrt2基因，所述的sgRNA为3或4条，至少3条sgRNA靶向的目标基因的外显子3，且外显子3上sgRNA靶向的靶位点之间间隔为10～50bp；较佳地15～40bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:83，SEQ ID NO:84，SEQ ID NO:85，SEQ ID NO:86；更佳地为：SEQ ID NO:84，SEQ ID NO:85，SEQ ID NO:86。
在另一优选例中，所述的待敲除的目标基因为ArntL基因，所述的sgRNA为3，4，5，6，7，8或9条，靶向的目标基因的外显子13，且外显子13上sgRNA靶向的靶位点之间间隔为10～80bp；较佳地40～70bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的3，4，5或6个：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；更佳地为：SEQ ID NO:92，SEQ ID NO:95，SEQ ID NO:98。
在另一优选例中，所述的待敲除的目标基因为Y染色体基因；较佳地为Zfy1；Ube1y1；Kdm5d；Eif2s3y；Ddx3y；Usp9y；Sry；Erdr1；
更优选地，所述的待敲除的目标基因为Zfy1，所述的sgRNA靶向的目标基因上的 靶位点为SEQ ID NO:67，SEQ ID NO:68；或
所述的待敲除的目标基因为Ube1y1，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:69，SEQ ID NO:70；或
所述的待敲除的目标基因为Kdm5d，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:71，SEQ ID NO:72；或
所述的待敲除的目标基因为Eif2s3y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:73，SEQ ID NO:74；或
所述的待敲除的目标基因为Ddx3y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:75，SEQ ID NO:76；或
所述的待敲除的目标基因为Usp9y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:77，SEQ ID NO:78；或
所述的待敲除的目标基因为Sry，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:79，SEQ ID NO:80；或
所述的待敲除的目标基因为Erdr1，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:81，SEQ ID NO:82。
在另一优选例中，还提供前面任一所述的方法的用途，用于制备基因敲除的动物细胞、动物受精卵或动物，其中所述的动物是目标基因的基因功能完全敲除或大部分敲除的动物；所述的目标基因的基因功能大部分敲除的动物的体内，待敲除目标基因未发生有效敲除的细胞占总细胞数的20％以下；更佳的15％以下；更佳地10％以下；进一步更佳地5％以下。
在另一优选例中，所述的目标基因的基因功能完全敲除或大部分敲除的动物，为仅经历一个生育周期即获得的动物(即F0代的动物)。
在另一优选例中，还用于进行动物基因功能研究；或用于制备目标基因的基因功能完全敲除或大部分敲除的动物，并将之用于基因功能研究，或将之用于胚胎发育研究。较佳地，该用途为非诊断或治疗性的用途。
在本发明的另一方面，提供用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为GFP基因，所述的sgRNA为2，3或4条，各sgRNA靶向的目标基因上的靶位点之间间隔为30～200bp；较佳地40～150bp；更佳地50～130bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的2，3或4个：SEQ ID NO:48，SEQ ID NO:49，SEQ ID NO:50，SEQ ID NO:51。
在本发明的另一方面，提供用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Tyr基因，所述的sgRNA为3，4，5或6条，各sgRNA靶向的目标基因的外显子 4及任选地与该外显子邻近的内含子，且sgRNA靶向的靶位点之间间隔为10～100bp；较佳地10～70bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的3，4，5或6个：SEQ ID NO:52，SEQ ID NO:53，SEQ ID NO:54，SEQ ID NO:55，SEQ ID NO:56，SEQ ID NO:57；更佳地为选自下组的3或4个：SEQ ID NO:53，SEQ ID NO:54，SEQ ID NO:55，SEQ ID NO:56。
在本发明的另一方面，提供用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Tet1基因，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子2，且各sgRNA靶向的靶位点之间间隔为80～200bp；较佳地100～190bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:58，SEQ ID NO:59，SEQ ID NO:60。
在本发明的另一方面，提供用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Tet2基因，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子3，且各sgRNA靶向的靶位点之间间隔为100～150bp；较佳地110～135bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:61，SEQ ID NO:62，SEQ ID NO:63。
在本发明的另一方面，提供用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Tet3基因，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子4，且各sgRNA靶向的靶位点之间间隔为180～280bp；较佳地190～270bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:64，SEQ ID NO:65，SEQ ID NO:66。
在本发明的另一方面，提供用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Prrt2基因，所述的sgRNA为3或4条，至少3条sgRNA靶向的目标基因的外显子3，且外显子3上sgRNA靶向的靶位点之间间隔为10～50bp；较佳地15～40bp；更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:83，SEQ ID NO:84，SEQ ID NO:85，SEQ ID NO:86；更佳地为：SEQ ID NO:84，SEQ ID NO:85，SEQ ID NO:86。
在本发明的另一方面，提供用于制备目标基因的基因功能完全敲除或大部分敲除基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为ArntL基因，所述的sgRNA为3，4，5，6，7，8或9条，靶向的目标基因的外显子13，且外显子13上sgRNA靶向的靶位点之间间隔为10～80bp；较佳地40～70bp；更 优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的3，4，5或6个：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；更佳地为：SEQ ID NO:92，SEQ ID NO:95，SEQ ID NO:98。
在本发明的另一方面，提供用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Y染色体基因Zfy1，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:67，SEQ ID NO:68；或
该sgRNA用于敲除的目标基因为Y染色体基因Ube1y1，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:69，SEQ ID NO:70；或
该sgRNA用于敲除的目标基因为Y染色体基因Kdm5d，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:71，SEQ ID NO:72；或
该sgRNA用于敲除的目标基因为Y染色体基因Eif2s3y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:73，SEQ ID NO:74；或
该sgRNA用于敲除的目标基因为Y染色体基因Ddx3y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:75，SEQ ID NO:76；或
该sgRNA用于敲除的目标基因为Y染色体基因Usp9y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:77，SEQ ID NO:78；或
该sgRNA用于敲除的目标基因为Y染色体基因Sry，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:79，SEQ ID NO:80；或
该sgRNA用于敲除的目标基因为Y染色体基因Erdr1，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:81，SEQ ID NO:82。
在本发明的另一方面，提供用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的试剂盒，所述试剂盒中包含前面任一所述的用于制备目标基因的基因功能完全敲除或大部分敲除的动物的sgRNA，或能形成所述sgRNA的核酸。
在一个优选例中，所述的试剂盒中还包括：Cas9 mRNA或能形成所述Cas9 mRNA的核酸；或使用说明书。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、用C-CRISPR在GFP胚胎中完全敲除GFP基因。
(A)位于GFP位点的sgRNA靶点和实验设计的示意图。Cas9 mRNA和单个或多个靶向GFP的sgRNA共同注射至单个小鼠受精卵中，然后在囊胚阶段观察其GFP信号。GFP IF和GFP IR：GFP基因型鉴定的内部引物；GFP OF和GFP OR：GFP基因型鉴定 的外部引物。图中
Figure PCTCN2018081016-appb-000001
表示相邻sgRNA靶向的靶位点之间间隔有100bp的碱基。后图中，
Figure PCTCN2018081016-appb-000002
均表示相邻sgRNA靶向的靶位点之间间隔有x(为一具体数字)bp的碱基。
(B)各种GFP靶向方式导致的囊胚中GFP信号图。在第一张图中，GFP阴性囊胚(绿色箭头，在囊胚中任何细胞中都没有GFP信号)，GFP阳性的嵌合囊胚(红色箭头，在囊胚中部分细胞有GFP信号)。
(C)各种GFP靶向方式产生的GFP阴性囊胚(无嵌合)的比例柱状图。两个及两个以上sgRNA靶向(sgRNA-GFP-A+B，A+B+C或A+B+C+D)比单个sgRNA靶向(sgRNA-GFP-A,B,C,D)产生更高比例的GFP阴性囊胚。条上的数字代表所统计的囊胚总数(***P<0.001，卡方检验)。
(D)各种GFP靶向方式产生的胚胎囊胚率。数字代表注射的受精卵总数。
(E)GFP被编辑的囊胚的基因型鉴定。被标记了*的囊胚的PCR产物进一步送去测序。所有的被基因型鉴定过的囊胚没有GFP信号，有小的插入删除(比如，sgGFP-A+B#1)，大片段外显子删除(比如，sgGFP-A+B#16)或两者皆有(比如，sgGFP-A+B#5)。NC，阴性对照。
(F)以上被标记了*的囊胚的DNA序列。
(G)GFP被编辑的囊胚中大片段外显子删除(LED)的频率；数字代表所统计的囊胚总数。
图2、一步法用C-CRISPR获得无嵌合的Tyr敲除小鼠。
(A)位于Tyr位点的sgRNA靶点的示意图。Cas9 mRNA和单个或多个靶向Tyr的sgRNA共同注射至单个小鼠受精卵中，然后移植受体。Tyr IF和Tyr IR：Tyr基因型鉴定的内部引物；Tyr OF和Tyr OR：Tyr基因型鉴定的外部引物。
(B)不同Tyr靶向策略导致的小鼠色素沉着表型的示意图。WT，具有完全色素沉着的野生型小鼠；Albino，没有色素的小鼠；嵌合体，有嵌合状色素沉着的小鼠。
(C)Tyr靶向获得的小鼠色素沉着表型的代表性结果。绿色箭头：Albino白化；红色箭头：嵌合状色素沉着。
(D)Tyr靶向产生的白化小鼠的比例柱状图。两个及两个以上sgRNA靶向(sgRNA-Tyr-B+C，B+C+D，B+C+D+E，A+C+F或A+C+D+F)比单个sgRNA靶向(sgRNA-Tyr-C或D)产生更高比例的白化小鼠比例。数字代表所统计的小鼠总数(***P<0.001，卡方检验)。
(E)Tyr靶向获得的小鼠出生率。数字代表移植的胚胎总数。
图3、Tyr基因编辑小鼠的DNA序列分析。
(A)实验设计示意图。小鼠尾部、整个囊胚或者注射胚胎的八细胞期单个卵裂球均被用于基因型分析。
(B)由sgRNA-Tyr-B+C+D+E靶向产生的白化小鼠的尾巴进行测序鉴定。#2小鼠有大片段外显子删除和插入删除。LED，大片段外显子删除；Indel，插入或删除碱基。每个柱子上面的数字：测序的TA克隆总数。
(C)Tyr靶向导致的不同突变类型的囊胚比例。数字代表测序和分析的TA克隆总数。WT，野生型等位基因。
(D)Tyr靶向导致的不同基因型的囊胚比例。纯KO：拥有完全敲除突变的囊胚。嵌合体：同时具有野生型等位基因和敲除突变的囊胚。数字代表统计的囊胚总数。
(E&F)由sgRNA-Tyr-C(E)和sgRNA-Tyr-B+C+D+E(F)靶向产生的16细胞胚胎中单个卵裂球的代表性序列。每个胚胎大约有50％的卵裂球被成功扩增和测序。sgRNA靶向的序列被用绿色标记，而PAM序列被用红色标记；被删除的核苷酸用连字号表示，而虚线表示的是被省略的区域。
(G&H)Tyr靶向导致的8至16细胞胚胎的单个卵裂球的不同突变类型(G)和基因型(H)的比例。LED，大片段外显子删除；Bi-allelic，双等位基因突变；Mono-allelic，单等位基因突变。数字代表所分析等位基因或卵裂球的总数。
(I)Tyr靶向导致的8至16细胞胚胎的不同突变型比例。数字代表所分析的所有胚胎的总数。
图4、一步法用C-CRISPR获得无嵌合的三Tet基因敲除小鼠。
(A)针对Tet1、Tet2和Tet3位点设计的sgRNA示意图，每个位点有三个sgRNA。
(B)完整的Tet基因编辑的E7.5天胚胎和对照胚胎的比例。数字代表所统计的胚胎总数(***P<0.001，**P<0.01，卡方检验)。
(C)通过对于组织切片进行免疫染色来观察不同Tet靶向策略导致的E7.5天胚胎中的5hmC(红)和5mC(绿)的水平。白色虚线：E7.5天胚胎的外胚层，右边框中的区域是更高清晰度的图像。“5hmC”，5-羟甲基胞嘧啶；“5mC”，5-胞嘧啶；Bar：50μm。
(D)在E7.5天胚胎中的5mC和5hmC的相对比例。每个数据点代表了从一张组织切片中测量出来的平均荧光强度的比例(5hmC/5mC)。误差线，标准差(***P<0.001，**P<0.01，非配对t-test)。
(E)不同Tet基因靶向形式获得的小鼠和对照小鼠的出生率。数字表示所有移植入代孕母鼠中的胚胎总数(***P<0.001，卡方检验)。
图5、用C-CRISPR敲除单个Y染色体基因的F0表型分析结果。
(A)位于Y染色体的八个靶向基因示意图。每个基因设计了两个sgRNA。红色代表之前研究中用传统方法敲除小鼠的基因；绿色代表的是同时位于X和Y染色体的基因。
(B)用C-CRISPR方法敲除单个Y染色体基因所获得的小鼠出生率。数字代表总的移植胚胎数。Erdr1基因靶向导致胚胎致死。
(C)删除了不同的Y染色体基因后的小鼠性别比例。
(D至F)敲除不同Y染色体基因的小鼠体内睾丸的占体重的比例(D)，精子浓度(E)和前向运动精子比例(F)。数字代表统计过的样本数目(***P<0.001，**P<0.01，*P<0.05，卡方检验)。
(G)删除了不同Y染色体基因的成体小鼠的睾丸切片的柱状分析。箭号，曲精小管中不正常的空泡；箭头，精子。不正常只有在Eif2s3y敲除的睾丸中被发现。Bar：100μm。
图6、一步法用C-CRISPR获得双等位基因Prrt2突变的食蟹猴。
(A)Prrt2位点的sgRNA靶向位置的示意图。
(B)从sgRNA-Prrt2-C or sgRNA-Prrt2-B+C+D靶向的八细胞胚胎的单个卵裂球中扩增出包括靶向位点的PCR产物，并进行Sanger测序。sgRNA靶向的序列被用绿色标记，而PAM序列被用红色标记；被删除的核苷酸用连字号表示，而虚线表示的是被省略的区域。
(C&D)Prrt2靶向获得的八细胞胚胎的单个卵裂球的不同突变类型(C)和基因型(D)的比例。LED，大片段外显子删除。
(E)Prrt2靶向获得的整个八细胞胚胎的基因型的比例。
(F)Prrt2靶向获得流产和存活的猴子中尾部、耳朵和血细胞中不同突变类型的比例。由sgRNA-Prrt2-A靶向获得流产猴子：猴#2、#3；由sgRNA-Prrt2-A靶向获得存活猴子：猴#4、#8、#10；由sgRNA-Prrt2-B+C+D靶向获得存活猴子：猴#11、#12。
(G)两只存活的带双等位基因突变猴子(#11、#12)的血细胞和成纤维细胞的单细胞分析。数字代表被分析的细胞总数。
(H)拥有类似PDK行为的Prrt2敲除猴子的照片。
(I)蛋白质免疫印迹法检测一个流产的Prrt2编辑猴子(#3)和对照野生型猴子(#6)的多个样本中皮层和小脑的Prrt2表达。值得注意的是，猴子#3的小脑中Prrt2表达被明显降低，但并非完全去除。
图7、Cas9 mRNA和sgRNA共注至小鼠MII卵母细胞后获得的小鼠嵌合率。
(A)注射过程示意图。
(B)在注射至MII卵母细胞后mCherry的表达情况。在注射的4小时后观察到红色信号，而且能保持24小时。
(C)针对Tet1、Tet2位点设计的sgRNA示意图。Tet1或Tet2IF和IR：Tet1、Tet2基因型鉴定的内部引物；Tet1或Tet2OF和OR：Tet1、Tet2基因型鉴定的外部引物。
(D)通过一步法和两步法注射获得的嵌合胚胎的代表性序列。sgRNA靶向的序列被用绿色标记，而PAM序列被用红色标记；被删除的核苷酸用连字号表示。
(E)根据DNA测序结果判定的通过一步法和两步法获得的有嵌合的囊胚比例柱状图。在线上的数字，表示测序的囊胚总数。
(F)通过一步法和两步法注射获得的胚胎囊胚率。数字代表注射的胚胎总数。两步 法注射的囊胚率比一步法要低(***P<0.001，卡方检验)。
(G)被编辑的等位基因相位特征。
(H)在靶向位点发现的插入删除的大小分布。
图8、靶向基因Tyr获得的小鼠尾巴、囊胚和卵裂的具有代表性的PCR产物和序列。
(A)sgRNA-Tyr-B+C+D+E靶向获得的九只小鼠的PCR产物。小鼠#1到#6的PCR产物均进行TA克隆连接并且测序。标记有*的尾巴的PCR产物的测序结果见图8B。NC，阴性对照。
(B)sgRNA-Tyr-B+C+D+E靶向获得小鼠#5尾巴的代表性序列。sgRNA靶向的序列被用绿色标记，而PAM序列被用红色标记；被删除的核苷酸用连字号表示，而虚线表示的是被省略的区域。
(C)sgRNA-Tyr-C或sgRNA-Tyr-B+C+D+E靶向获得的囊胚的具有代表性的PCR产物。标记有*的囊胚的PCR产物做了进一步测序，见图2D和2E。
(D&E)sgRNA-Tyr-C靶向获得的囊胚#1(D)和sgRNA-Tyr-B+C+D+E靶向获得的囊胚#5(E)的代表性序列。
(F)在靶向位置的插入删除和大片段外显子缺失的大小分布。
(G)sgRNA-Tyr-C或sgRNA-Tyr-B+C+D+E靶向获得卵裂球的具有代表性的PCR产物。
(H)靶向基因Tyr获得胚胎中成功扩增出的卵裂球比例。数字代表从靶向基因Tyr获得胚胎中分离出的卵裂球总数。
图9、靶向基因Tyr获得的两细胞胚胎的DNA测序分析结果。
(A)实验设计。两细胞注射胚胎的单个卵裂球被用于基因型鉴定。
(B)Tyr基因编辑后的胚胎中成功扩增出的卵裂球比例。数字代表从靶向基因Tyr获得胚胎中分离出的卵裂球总数。
(C)靶向基因Tyr获得的两细胞胚胎中不同基因型的比例。纯KO：发生完全敲除突变的两细胞胚胎；嵌合体：既含有野生型等位基因也含有敲除突变的两细胞胚胎。数字代表所统计的两细胞胚胎总数。
(D&E)Tyr靶向导致的两细胞胚胎的单个卵裂球的不同突变类型(G)和基因型(H)的比例。LED，大片段外显子删除；Bi-allelic，双等位基因突变；Mono-allelic，单等位基因突变。数字代表所分析等位基因或卵裂球的总数。
(F&G)sgRNA-Tyr-C(F)或sgRNA-Tyr-B+C+D+E(G)靶向获得的一个两细胞胚胎中单个卵裂球的代表性序列。sgRNA靶向的序列被用绿色标记，而PAM序列被用红色标记；被删除的核苷酸用连字号表示，而虚线表示的是被省略的区域。
图10、用C-CRISPR方法编辑Y染色体基因所获得的小鼠的DNA序列分析。测序了Y染色体上不同基因被进行了靶向所获得小鼠的尾巴DNA。LED，大片段外显子删 除；Indel：插入或删除碱基；每个柱子上的数字：测序的TA克隆总数。
图11、靶向基因Prrt2获得的猴子卵裂球、囊胚和组织的具有代表性的PCR产物和序列。
(A)用于Prrt2靶向的每个sgRNA的切割效率。Cas9 mRNA和sgRNA共注至受精卵中，然后培养至囊胚再进行基因型鉴定。数字代表用于基因型鉴定的囊胚总数。
(B)从sgRNA-Prrt2-C靶向获得的胚胎#4和sgRNA-Prrt2-B+C+D靶向获得的胚胎#3的卵裂球中扩增出的PCR产物。卵裂球的PCR产物都进行了TA克隆并且测序。
(C)sgRNA-Prrt2-B+C+D靶向获得的猴#11和#12的耳朵、尾巴和血细胞中DNA序列代表性结果。sgRNA靶向的序列被用绿色标记，而PAM序列被用红色标记；被删除的核苷酸用连字号表示，而虚线表示的是被省略的区域。
(D&E)猴#11和#12的单个成纤维细胞和血细胞的具有代表性的PCR产物和序列。被标记了*的单个细胞的PCR产物进一步测序，并在右列展示。
图12、利用C-CRISPR一步法在猴子胚胎中做到Arntl基因完全敲除。
(A)针对Arntl位点设计的sgRNA示意图。
(B)用于Arntl靶向的每个sgRNA的切割效率。Cas9 mRNA和sgRNA(Arntl-1至6)共注至受精卵中，然后培养至囊胚再进行基因型鉴定。数字代表用于基因型鉴定的囊胚总数。
(C)在COS-7细胞中进行sgRNA靶向示意图。带有Cas9、sgRNA和mCherry转入COS-7细胞中后，分选出mCherry阳性细胞，然后用于T7E1分析。
(D)Arntl靶向的T7E1分析。箭头表示的是T7E1产物作用的位置。对照是正常的COS-7细胞基因组DNA。
(E)Arntl-X+X+X靶向获得的八细胞胚胎被测序。LED，大片段外显子删除；Indels:插入或删除碱基。每个柱子上的数字：测序的TA克隆总数。
(F)在靶向位点发现的插入删除和大片段外显子删除的大小分布。
(G)Arntl-X+X+X靶向获得的八细胞胚胎中具有代表性的序列。sgRNA靶向的序列被用绿色标记，而PAM序列被用红色标记；被删除的核苷酸用连字号表示，而虚线表示的是被省略的区域。
图13、分析Tyr靶向获得的小鼠和Prrt2靶向获得猴子中的脱靶效应
(A)sgRNA-Tyr-B+C+D+E靶向获得的六只小鼠被用于脱靶分析。本发明人针对每个sgRNA选在了多达10个可能发生的突变位点。并从这些基因组位点处扩增出PCR产物，进行TA克隆连接，并且进行测序。红色：与靶向序列不配对。
(B)sgRNA-Prrt2-B+C+D靶向获得的两只活着的猴子(#11和#12)被用于脱靶分析。本发明人针对每个sgRNA选择了多达三个碱基不匹配的脱靶位点进行检测。红色：与靶向序列不配对。
具体实施方式
本发明人经过深入的地研究，首次揭示一种鸡尾酒式CRISPR/Cas9系统及利用该系统进行制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物(无嵌合或低嵌合率的基因敲除动物)的方法。
如本文所用，所述“动物(mammal)”是指哺乳纲的动物，包含人、非人灵长类动物(猴、猩猩)、家畜与农畜(例如，猪、绵羊、牛)，鼠(小鼠)，以及啮齿动物(例如，小鼠、大鼠、兔)。基因功能完全敲除动物个体内，每个细胞基因功能被破坏，但每个细胞基因型不一定完全一致。
如本文所用，所述的“目标基因”是指动物基因组中需要进行敲除操作的感兴趣的基因。
如本文所用，所述的“目标基因上的靶位点”是指“目标基因”中的一个片段，基于该“目标基因上的靶位点”设计的sgRNA可识别该靶位点，由此在该位置发生Cas9编码的蛋白的切割。所述的“目标基因上的靶位点”的长度为18-26个核苷酸。
如本文所用，所述的“sgRNA”即“单独导向RNA(Single-guide RNA，sgRNA)”或“单导向RNA”，其是基于“目标基因上的靶位点”设计，其包含的序列足以与内切核酸酶Cas9协同作用，引导发生Cas9介导的靶位点上DNA双链断裂。
如本文所用，所述的“等位基因(allele)”是指在一对同源染色体上，占有相同座位的一对基因，它控制一对相对性状。当一个体的一基因有两个相同的等位基因，则称此个体就该基因或等位基因而言为纯合子。当一个体的一基因有两个不同的等位基因，则称此个体就该基因而言为杂合子的。
如本文所用，所述的“低嵌合率的基因敲除动物”是一种动物，其体内待敲除目标基因未发生有效敲除的细胞占总细胞数的20％以下；更佳的15％以下；更佳地10％以下；进一步更佳地5％以下。更特别地，本发明中，所述的“低嵌合率的基因敲除动物”为仅经历一个生育周期即获得的动物，即F0代的动物。
如本文所用，除非另外说明，所述的“大片段缺失”是指在进行如本发明所述的基因编辑操作后，目标基因中存在大于或等于30bp的连续的碱基缺失。
如本文所用，除非另外说明，所述的“插入缺失”是指在进行如本发明所述的基因编辑操作后，目标基因中存在小于30bp的连续的碱基缺失。
如本文所用，“鸡尾酒式CRISPR/Cas9系统(Cocktail of CRISPR/Cas9 system)”，简称为“C-CRISPR”，即基于CRISPR/Cas9技术，通过将Cas9 mRNA以及两个或多个sgRNA的鸡尾酒式混合物引入到动物细胞(特别是动物受精卵细胞)，来获得高效基因敲除效率的方法。
基因编辑方法
CRISPR/Cas9系统是一种非常高效的基因编辑方法，但是大部分被基因编辑过的动物都有嵌合性，也就是说它们只有一部分细胞的基因发生了编辑。本发明人发现，借由向动物细胞(特别是受精卵)内引入Cas9 mRNA和多个针对于每个基因的关键外显子的毗连(一般地，间隔为10-300bp)的单独导向RNA(Single-guide RNA，sgRNA)，可以在动物胚胎中，达到对一个或多个基因高达100％的敲除率。
因此，本发明提供了一种制备基因敲除的动物细胞的方法，所述方法包括：(1)根据待敲除的目标基因的核酸序列，制备两条或多条靶向目标基因上不同靶位点的sgRNA；和(2)将(1)的sgRNA或能形成所述sgRNA的核酸(例如，DNA)、Cas9 mRNA或能形成所述Cas9 mRNA的核酸(例如，DNA)共转入动物细胞中，获得基因敲除的动物细胞。
本发明也提供了制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的方法，所述方法包括：(1)根据待敲除的目标基因的核酸序列，制备两条或多条靶向目标基因上不同靶位点的sgRNA；(2)将(1)的sgRNA或能形成所述sgRNA的核酸(如DNA)、Cas9 mRNA或能形成所述Cas9 mRNA的核酸(如DNA)共转入受精卵中，获得基因敲除的动物受精卵；(3)使(2)的受精卵发育产生所述的目标基因的基因功能完全敲除或大部分敲除的基因敲除动物。
本发明的方法中，所述的动物细胞为动物受精卵，所述的受精卵可发育为目标基因的基因功能完全敲除或大部分敲除的基因敲除动物。
合适的sgRNA靶位点，会带来更高的基因编辑效率，所以在着手进行基因编辑前，设计并找到合适的靶位点至关重要。尽管sgRNA的制备是本领域已知的技术，目前也已有一些软件可用于sgRNA的辅助设计，但是选择合适的靶位点仍然是至关重要的，且是仅凭借软件分析难以做到的。在设计特异性靶位点后，还需要进行体外细胞活性筛选，以获得有效的靶位点用于后续实验。
作为本发明的优选方式，在设计针对两条或多条靶向目标基因上不同靶位点的sgRNA时，所述的sgRNA靶向的目标基因上的靶位点之间间隔为9～500bp是较为合适的。较佳地，间隔可以为10～300bp；例如，15bp，20bp，30bp，40bp，50bp，60bp，70bp，80bp，90bp，100bp，120bp，150bp，180bp，200bp，250bp，280bp。应理解，根据基因的不同情形，外显子的不同长度及不同分布，可能还存在其他的选择，本领域技术人员可以依据本发明所给出的技术方案来获得合适的选择。
在设计针对两条或多条靶向目标基因上不同靶位点的sgRNA时，sgRNA的数量一般在3～30条之间，例如可以是4，5，6，7，8，9，10，12，15，18，20，25条。
作为本发明的优选方式，所述的多条靶向目标基因上不同靶位点的sgRNA，其 中至少2条、较佳地至少3条所靶向的目标基因上的靶位点位于同一外显子区域。较佳地，该外显子是该目标基因的关键外显子，其被编辑后使得基因功能发生显著性变化或丧失功能。
作为本发明的优选方式，所述的多条靶向目标基因上不同靶位点的sgRNA，能够在其所靶向的目标基因的靶位点区域引入移码突变。较佳地，在目标基因的功能域或编码区内部设计sgRNA有助于引入移码突变。
作为本发明的优选方式，所述的多条靶向目标基因上不同靶位点的sgRNA，能够在其所靶向的目标基因的靶位点区域引入大片段缺失。较佳地，该大片段缺失为外显子区域的大片段缺失。本发明人发现，相对短距离毗邻的sgRNA可以带来高频的外显子大片段缺失而非普通的插入缺失，这会引起更高概率的全基因删除，更有效地提高基因敲除的效率。
作为本发明的优选方式，所述的多条靶向目标基因上不同靶位点的sgRNA，能够在所靶向的目标基因的靶位点区域同时引入插入缺失及大片段缺失。
要确保高效的基因敲除，本发明的C-CRISPR方法较佳地满足以下的一个或多个条件：(a)使用多个sgRNA靶向基因的一个外显子；(b)多个sgRNA靶向外显子上比较靠近的几个位点(间隔如前所述)；(3)靶向基因的关键外显子，有时候在蛋白质的关键结构域上，只需一两个氨基酸突变就能彻底扰乱蛋白质的功能；(4)预先在胚胎中检测sgRNA的切割效率，尤其是针对猴子的基因编辑，预先筛选效率高的sgRNA。这些条件可以促使在一般的插入缺失之外还能产生外显子大片段删除，继而令基因得以完全敲除。
在确定了靶位点之后，可以采用已知的方法来使得sgRNA及Cas9被引入到细胞内。一些可以考虑的引入细胞的方式是：瞬时表达，如转染、电穿孔，或非整合型病毒(AAV或腺病毒)等。当应用于受精卵细胞时，较为优选的是采用显微注射来使得sgRNA及Cas9被引入到细胞内。作为一种选择，所述的能形成所述sgRNA的核酸为核酸构建体或表达载体，或所述的能形成所述Cas9 mRNA的核酸为核酸构建体或表达载体，将这些表达载体导入到细胞内，从而在细胞内形成有活性的sgRNA及Cas9mRNA。作为更优选的选择，可以体外转录获得携带有启动子的Cas9 mRNA以及携带有启动子的sgRNA，将它们注射入细胞内。所述的启动子包括但不限于T7启动子，U6启动子。
用途
本发明所述的方法可用于制备基因敲除的动物细胞、动物受精卵或动物，其中所述的动物是目标基因的基因功能完全敲除或大部分敲除的基因敲除动物；所述的低嵌合率的基因敲除动物的体内，待敲除目标基因未发生有效敲除的细胞占总细胞数的 20％以下；更佳的15％以下；更佳地10％以下；进一步更佳地5％以下。所述的目标基因的基因功能完全敲除或大部分敲除的基因敲除动物，为仅经历一个生育周期即获得的动物。
利用本发明所述的方法获得的目标基因的基因功能完全敲除或大部分敲除的基因敲除动物，可用于进行动物基因功能研究，了解动物生长发育的各个环节、各种状态下感兴趣的基因的功能，所参与的生物调控机制、信号通路。本发明所述的方法可用于进行动物胚胎发育研究，了解胚胎发育过程中感兴趣的基因的功能。
本发明的方法可应用于靶向多种感兴趣的、需要进行功能研究的基因，包括但不限于：报告基因(比如荧光蛋白基因)、表型标记基因、结构基因、功能性基因等进行基因敲除，获得F0代的非嵌合或嵌合基因敲除动物。作为本发明的优选方式，所述的基因包括但不限于：GFP，Tyr，Tet1，Tet2，Tet3，Prrt2，ArntL，Y染色体基因(如Zfy1，Ube1y1，Kdm5d，Eif2s3y，Ddx3y，Usp9y，Sry，Erdr1)。
CRISPR/Cas9系统是个很有效的基因编辑方法，但是大部分基因编辑动物都表现出嵌合性，这妨碍了人们高效地对基因敲除进行表型分析。在本发明当中，使用C-CRISPR，可以让注射CRISPR/Cas9的动物将近100％地在所有细胞中都表现出高效地基因删除，无论删除的是单个基因还是多个基因。这将高度合乎伦理道德的观点，尤其是在使用非人灵长类作为动物模型研究的时候。
在本发明的具体实施例中，对分别敲除Y染色体上八个基因(如Zfy1，Ube1y1，Kdm5d，Eif2s3y，Ddx3y，Usp9y，Sry，Erdr1)的F0代小鼠的表型分析，充分表明了本发明的方法在制造基因敲除动物中的稳定性。重要的是，该方法能够做到高效的敲除整个基因。
在本发明的具体实施例中，针对表型标记基因进行了C-CRISPR方法敲除研究。表型标记基因例如：GFP的荧光，Tyr的皮肤白化，Tet-1、2、3的5’羟甲基化胞嘧啶作用，Sry的性别决定等。这些实施例的结果都表明了胚胎和动物中目标基因可发生完全敲除，可实现功能完全丧失。
在本发明的具体实施例中，还对基因编辑鼠、猴胚胎的单囊胚或单裂球基因型进行了分析，其中所有细胞都有目标基因的双等位基因突变，其中大部分(80％)都有外显子大片段删除，这在任何情况下都是基因完全删除的强力证据。外显子大片段删除较之常规的插入缺失，会大大增加基因功能丧失的可能性，因为这有可能产生没有移码突变但是功能丧失的基因产物。
在不少研究中获得了基因敲除猴子，但是这些猴子都有很高的嵌合现象。这个会导致这些基因编辑的猴子很难展现清晰的表型。现有技术中，要产生能用来做表型分析的猴子需要历经至少两个生育周期(12年或更久)。利用本发明的C-CRISPR方法，本发明人直接在F0代得到了非嵌合的基因编辑猴，从注射胚胎开始算的话本发明人 仅仅用了六个月，这充分说明了本发明的方法非常适用于快速建立基因编辑猴模型。
在本发明的具体实施例中，通过C-CRISPR方法敲除了Prrt2基因，一步到位没有嵌合地构建了人类阵发性非运动源性运动障碍(人类运动失调，PKD)的猴子模型。PKD被发现是一种单基因导致的神经疾病，根源是Prrt2基因的突变。构建PKD的猴子模型将有助于理解PKD的病理学机制并开发潜在的治疗手段。据本发明人所知，这是CRISPR技术第一次被成功用于获取有疾病行为表型的基因敲除猴。在此展现出的C-CRISPR之高效，使得在F0代猴子当中进行基因研究不再是梦想。
在本发明的具体实施例中，也对于那些多个sgRNA靶向的动物进行了全面的脱靶分析，没有发现明显的脱靶效应。
基因编辑试剂盒
本发明还提供了用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的试剂盒，所述的试剂盒中包含针对于感兴趣的基因的、应用于进行C-CRISPR方法操作的sgRNA及Cas 9 mRNA或能够在体内或体外形成该sgRNA及Cas 9 mRNA的试剂。
所述的感兴趣的基因包括：GFP，Tyr，Tet1，Tet2，Tet3，Prrt2，ArntL，，Zfy1，Ube1y1，Kdm5d，Eif2s3y，Ddx3y，Usp9y，Sry，Erdr1等。针对这些感兴趣的基因，本发明人经过深入的研究和筛选，分别获得了适合于实现高效敲除的sgRNAs，建立了C-CRISPR系统。
其它常用于进行转基因操作的试剂也可被包含在所述的试剂盒中，以方便本领域技术人员使用，例如显微注射用的试剂等。此外，所述试剂盒中还可包含有指导本领域技术人员操作的使用说明书。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
Cas9 mRNA和sgRNA的获得
利用PCR方法以引物Cas9F和R(正向：TAATACGACTCACTATAGGGAGATTTCAGGTTGGACCGGTG；反向：GACGTCAGCGTTCGAATTGC)扩增质粒px260(Addgene)上的Cas9编码区域，同时将T7启动子加入产物中。T7-Cas9PCR产物纯化后作为体外转录(IVT)为mRNA的模板，所使用的试剂盒为mMESSAGE mMACHINE T7ULTRA kit(Life Technologies)，从而获得Cas9 mRNA。
利用PCR方法使用列表中引物(表1)扩增质粒px330(获自Addgene)，同时将T7启动子加入sgRNA模板中。T7-sgRNA PCR产物纯化后作为体外转录(IVT)sgRNA的模板，所使用的试剂盒为MEGA shortscript T7kit(Life Technologies)，从而获得sgRNA。
Cas9 mRNA和sgRNA都用MEGA clear kit(Life Technologies)进行纯化，并且用无RNase的水进行洗脱。
在应用于注射时，将针对相应的基因设计的sgRNA，连同Cas9 mRNA，共同显微注射到受精卵中。
表1、制备用作sgRNA体内转录模板的引物
Figure PCTCN2018081016-appb-000003
Figure PCTCN2018081016-appb-000004
表2、sgRNA靶序列
Figure PCTCN2018081016-appb-000005
Figure PCTCN2018081016-appb-000006
受精卵注射、胚胎培养和胚胎移植
对小鼠进行基因编辑时，首先激素超排C57BL/6雌鼠(三周大)或B6D2F1(C57BL/6X DBA2J)雌鼠(7-8周大)，然后分别与C57BL/6公鼠或者B6D2F1公鼠交配，然后从输卵管中收集受精卵。对于GFP基因的编辑，将纯合的Actin-GFP转基因公鼠(Okabe，M.,et al.(1997).FEBS letters 407，313-319)与野生型的母鼠交配后收集受精卵。Cas9mRNA(50μg/μl)和sgRNA(50μg/μl)混合后，用FemtoJet微量注射仪(Eppendorf)的持续水流模式注入到可以明显看到原核的受精卵胞质中，注射的过程在含5μg/ml细胞松弛素(CB)的HEPES-CZB培养基中完成，注射完后的胚胎在37℃、5％CO2的情况下在含氨基酸的KSOM培养基中养至1.5天的两细胞胚胎。随后，25-30枚两细胞胚胎移植入0.5天的假孕ICR母鼠的输卵管中。
对猴子进行基因编辑时，利用腹腔镜进行卵母细胞收集。在hCG刺激的32-36小时 后，从2-8mm直径的卵泡中吸出卵母细胞，收集到的卵母细胞培养在预平衡好的成熟培养基中。阻滞在减数分裂中期II的卵母细胞被用于胞浆内单精注射(ICSI)，然后观察两个原核的出现来判断其是否受精。受精卵中注射Cas9 mRNA(100μg/μl)和sgRNA(50μg/μl)。在注射后，胚胎放置在HECM-9培养基中培养，直到第二天移入假孕母亲体内。一些胚胎培养至桑葚或囊胚阶段，然后收集抽取基因组并且分析。
单细胞PCR分析
在体视显微镜下用玻璃管收集和转移单个细胞。小鼠或猴的8-16细胞阶段的胚胎用台式酸消化去除透明带，然后将胚胎转移至0.25％的胰酶中，温和的吹打来分离出单个卵裂球。最后卵裂球在0.25％的胰酶中洗7至10次，再转移至一个PCR管中。成纤维细胞或者白细胞逐步用KSOM稀释直到细胞完全分散。在KSOM中洗7到10次后，单个细胞转移至一个PCR管中。1.5μl裂解液(0.1％吐温20、0.1％TritonX-100和4μg/ml蛋白酶K)加入PCR管中，然后离心促进混合。混合物在56℃中反应30分钟，随后95℃反应5分钟。裂解产物被用于下一步巢式PCR的模板。
基因型分析
通过抽提尾巴组织的基因组DNA随后进行PCR反应来检测突变小鼠的基因型(用于检测的引物如表3)。ExTaq在95℃、3分钟的条件下被激活，然后PCR的程序为95℃30s，62℃30s和72℃1分钟，这样反复34个循环后，最终72℃延伸5分钟。PCR产物经过割胶纯化并且DNA测序来分析。对于囊胚，KSOM清洗6次后，单个囊胚移入含有1.5μl裂解液(0.1％吐温20、0.1％TritonX-100和4μg/ml蛋白酶K)的PCR管中，然后在56℃中反应30分钟，随后95℃反应10分钟以失活蛋白酶K。用巢式引物来进行PCR扩增。ExTaq在95℃、3分钟的条件下被激活，然后PCR的程序为95℃30s，62℃30s和72℃1分钟，这样反复34个循环后，最终72℃延伸5分钟。第二轮PCR反应用上一轮的0.5μl PCR产物为模板，巢式内部引物为PCR引物进行反应。PCR反应的体系同前。
表3、用于基因型分析的引物
Figure PCTCN2018081016-appb-000007
Figure PCTCN2018081016-appb-000008
Figure PCTCN2018081016-appb-000009
HE染色和免疫染色
关于睾丸分析，部分睾丸用bouin’s溶液固定过夜。固定后的组织用石蜡进行包埋，然后用Microtome切成5μm厚度。在脱蜡后，复水的切片用苏木精-伊红进行染色。关于5hmC染色，在特定的天数时着床后胚胎从怀孕母鼠中剖出，然后用4％多聚甲醛进行固定，并且用OCT(Sakura)进行包埋，并且切成8μm厚度。用PBS清洗后，切片用盐酸溶液(4N盐酸，0.1％TritonX-100在蒸馏水中)处理，随后用PBS清洗，并且用封闭液(1％BSA，10％羊血清和含0.3％TritonX-100的PBS)进行通透。接下来，切片用一抗(小鼠抗5mC抗体，1:500，Eurogentec#BI-MECY-0100；兔抗5hmC抗体，1:1000，Active Motif#39792)4℃孵育过夜，二抗室温下孵育一小时。最后这些切片用抗淬灭剂(Invitrogen)进行封片，用LEICA TCS SP5 II共聚焦显微镜进行观察拍照。信号的强度用Leica Application Suite软件来测定。
使用计算机辅助手段的精子分析
缓冲液配方：NaCl 7.0g/L，KCl 149mg/L，CaCl 2.2H 2O 147mg/L，NaHCO 3 2.1g/L，MgSO 4.7H 2O 296mg/L，NaH 2PO 4-H 2O 49.7mg/L，Glucose 1g/L，Na pyruvate 121mg/L，Sucrose 6.63g/L，TAPSO(buffer)2.59g/L(10mM)，Penicilline 57mg/L，Streptomycine 29mg/L，pH＝7.3～7.4。精子用该缓冲液稀释至3-6×10 6个/ml，然后在37℃的皿中孵育20分钟。精子悬浮物在测定活力前先温和的混匀。每次活力测定时，用大孔径的枪头通过毛细现象吸取一滴5μl的精子悬浮液加入到一个事先预热好的Lejia载玻片的数数的卡槽中(深度，10μm)，然后用计算机辅助的精液分析(CASA)仪器(HTM-TOX IVOS精子活力分析仪，Animal Motility，version 12.3A；Hamilton Thorne Research)。放大倍数为10×。所有的样品都至少统计两次来避免因错误的取样所导致的误差。每个样品至少记录五个区域和至少100个有活力的精子。图像保存并进行后续分析。
猴子的行为学分析
Prrt2突变猴子的建群者(F0)自他们出生起开始进行行为观察。典型的异常特征用 摄像机(Sony HDR-XR520Handicam)记录。每个猴子分别被放在一个平台或者观察箱(1.5x 1x 1.1m)中根据不同的观察目的来进行记录。
蛋白免疫印迹法
从猴子死胎中取出大脑组织，然后用添加了1mM PMSF和蛋白酶抑制剂鸡尾酒(CST)的RIPA裂解液(50mM Tris–HCl(pH 7.4)，150mM NaCl，1％Triton X-100，1％sodium orthovanadate，1mM EDTA and 0.1％SDS)进行裂解。随后离心抽提蛋白，等量的蛋白在9％的SDS-聚丙烯酰胺胶上电泳，然后转移到聚偏二氟乙烯膜(Millipore)上，膜经过室温下在含5％脱脂奶粉的TBST里封闭一小时，然后用合适的一抗4℃孵育过夜。这里用的一抗有：兔抗PRRT2抗体(1:2000，Sigma)和HRP偶联的抗GAPDH抗体(1:8000，Kangchen)。第二天这些膜各自用TBST洗3次，每次10分钟，再用HRP偶联的抗兔二抗室温下两小时。再次清洗后，条带可以用ECL Plus Western Blot detection reagent(Tiangen)显色。
SURVEYOR分析法
Cos-7细胞在转染前24小时种到6孔板中。细胞在80-90％的时候用Lipofectamine 3000(Life Technologies)进行质粒转染。细胞瞬时用pX 330-mCherry-sgRNA质粒(基于addgene 42230建立))转染。转染后72小时，mCherry阳性细胞用流式细胞术分选出来，细胞重悬在裂解液(0.1％吐温20、0.1％TritonX-100和4μg/ml蛋白酶K)中，56℃中反应30分钟，随后95℃反应10分钟以失活蛋白酶K。SURVEYOR方法和之前报导的步骤一致。PCR扩增所使用的引物详见表3中。
全基因组测序和脱靶分析
全基因组测序是用Illumina HiSeq X Ten仪器来实现的，平均覆盖率分别为10倍(Tyr-B+C+D+E-#1，#2，#3，#4，#5，#6)和15倍(Prrt2-B+C+D-#11,#12)。合格的所测到的序列数据用BWA(v0.7.10)根据默认参数，定位到小鼠参考基因组(mm10)和拼接的食蟹猴基因组(v5)。去除那些测到的与所参考的基因组有小于50bp配对或者大于0错配的数据。sgRNA有可能造成脱靶的区域的预测方案跟之前报道的一样(Hsu et al.(2013).Nature biotechnology 31,827-832)，并且用CRISPR设计软件打分排位。本发明人对于每个sgRNA选取前20个脱靶位点，和60bp它们的相邻区域。然后将在这些区域的测序数据与参考序列进行BLAST比较，从而搜寻在样品里面因为脱靶效应造成的可能的插入删除模式。同样，也用PCR扩增了前10个最有可能脱靶位点的序列，并且进行DNA测序。扩增脱靶位点的引物详见表4。Illumina HiSeq序列数据已上传保存在NCBI，序列号为BioProjectID PRJNA352101。
表4、用于脱靶分析的引物
Figure PCTCN2018081016-appb-000010
Figure PCTCN2018081016-appb-000011
Figure PCTCN2018081016-appb-000012
Figure PCTCN2018081016-appb-000013
数据分析
所有P值都是根据t检验和卡方检验来分析获得。所有的误差线都代表标准差。
实施例1、完全删除转基因绿色荧光胚胎当中的绿色荧光基因
本发明人首先想知道在受精卵中提前注射Cas9 mRNA和sgRNA至小鼠MII卵中能否降低其嵌合现象。靶向的基因是Tet1，Tet2；应用于制作体内转录模板的引物如表1中相应序列(Tet1及Tet2及sgRNA-R)；sgRNA靶序列如表2中相应序列；应用于基因型分析的引物如表3中相应序列。两步法注射：注射Cas9 mRNA和sgRNA至小鼠MII卵母细胞，4个小时后注射精子至MII卵母细胞内。一步法注射：注射Cas9 mRNA和sgRNA至小鼠合子胚胎。结果，并未发现有明显的效果，嵌合现象没有明显降低，如图7A-F。
尽管本发明人调整设计了多种sgRNA一一实验，但效果仍然不理想。继而，本发明人试着使用多条而不是一条sgRNA，看看能不能降低CRISPR/Cas9编辑胚胎基因时的嵌合现象。为了能容易估计出嵌合率，本发明人使用纯合的Actin-EGFP雄性转基因小鼠(在全身广泛表达GFP)和野生型雌性小鼠配种得到受精卵，再向其中同时注入了Cas9 mRNA和靶向GFP的sgRNA，在囊胚阶段分析GFP表达(图1A)。本发明人设计了四个针对GFP同一外显子的sgRNA，这些sgRNA的靶序列彼此间隔10-200bp。应用于制作体内转录模板的引物如表1中相应序列(GFP-A、GFP-B、GFP-C、GFP-D)；sgRNA靶序列如表2中相应序列(GFP-A、GFP-B、GFP-C、GFP-D)；应用于基因型分析的引物如表3中相应序列(GFP)。
在注射大量胚胎之后，本发明人发现，只注射一个sgRNA的囊胚中有31-44％没有绿色荧光信号，其余的则表现为嵌合体，即其中一部分卵裂球依然有绿色荧光信号(图1B和1C)。作为对照，所有未经基因编辑而得到的囊胚都是GFP阳性的(图1B和1C)。
然而，注射多个(2，3或4)靶向GFP的sgRNA后，就无法在任何一个囊胚中检测到绿色荧光信号了(图1B和1C)。不仅如此，较之注射单个sgRNA，注射多个sgRNA 不会对胚胎发育表现出任何毒性(图1D)。继而，本发明人对通注射sgRNA所得的GFP阴性囊胚做了基因型检测，结果发现囊胚在GFP位点上表现出大片段缺失(认为>30bp或＝30bp)而非一般的插入缺失(认为<30bp)，而这个比例随着使用sgRNA数目增加而逐步增高(高达90％，图1E到1G)。
这些结果表明，对单个外显子使用两个及两个以上紧密毗连的sgRNA可以完全敲除单个等位基因，本发明人将这种方法命名为“鸡尾酒式CRISPR/Cas9系统”(Cocktail of CRISPR/Cas9 system，简称为C-CRISPR)。本发明人分析认为，完全删除基因能有如此之高效似乎是因为同时引入了移码突变和外显子删除。
实施例2、完全删除小鼠的Tyr基因
为了更进一步检测C-CRISPR是否可以完全删除内源性双等位基因，本发明人靶向了受精卵的Tyrosinase基因(Tyr，用于色素沉积)，并借由两细胞胚胎移植获得了基因编辑小鼠(图2A)。拥有一或二个野生型Tyr拷贝的小鼠会表现出完全着色，反之，两个等位基因全都失效的小鼠则是白化的，因此通过目测就可以看出小鼠的嵌合性。应用于制作体内转录模板的引物如表1中相应序列(Tyr-A～Tyr-F及sgRNA-R)；sgRNA靶序列如表2中相应序列。sgRNA靶位点A(Tyr-A)、F(Tyr-F)位于外显子4两端的紧挨外显子的内含子位置；sgRNA靶位点B(Tyr-B)、C(Tyr-C)、D(Tyr-D)、E(Tyr-E)位于外显子4。
本发明人将基因编辑实验分成五组并通过观察小鼠色素沉积现象来探究C-CRISPR的效率和潜在机制(图2B)。若用单个靶向Tyr基因外显子的sgRNA(组I，只有插入缺失)，有很高比例的小鼠(77％)为嵌合体(图2C和2D)，这与之前的报告相一致(Yen et al.，2014)。相对的，用两个sgRNA靶向一个外显子(组II，有插入缺失和外显子大片段删除)大大降低了嵌合体小鼠的比例(仅为26％)。在组III(只有外显子删除)中，两个sgRNA靶向于两个紧挨着外显子的内含子，这也导致了较高的嵌合小鼠比例(68％)(图2C和2D)。有趣的是，本发明人发现使用三或更多靶向一个外显子的sgRNA能够完全消除嵌合现象(0％，组IV)，这种使用多个sgRNA的方法可能在遇到某些外显子太小不足以容纳那么多sgRNA位点的情况时遇阻。为了探究针对这类短外显子的替代方案，本发明人又设计了一个用两个靶向毗邻外显子的sgRNA再加一个靶向外显子内部的sgRNA的方案(组V)，发现这个方案可以把嵌合体的比例下降到7-8％(图2C和2D)。
本发明人还比较了增加sgRNA对于小鼠出生率的影响，结果发现，增加靶向基因的sgRNA并不会减少小鼠的出生率(30-50％，图2E)。
与ICR小鼠(白化品系)交配后，四个sgRNA(sgRNA-Tyr-B+C+D+E)打靶的基因修饰小鼠生育能力正常，100％的后代都是白化的，说明这些小鼠生殖细胞内的Tyr基因都被完全的敲除了(表4)。
表4、C-CRISPR基因修饰的小鼠的生殖能力和遗传能力
Figure PCTCN2018081016-appb-000014
综上所述，C-CRISPR可在F0代中高效产生没有嵌合的基因敲除鼠，上述五组的实验数据说明，同时引入移码突变和外显子大片段删除可以带来高效的基因删除。
实施例3、C-CRISPR完全删除基因的机制
为了进一步阐明C-CRISPR的机制，本发明人对Tyr基因编辑的胚胎和小鼠做了DNA序列分析。对6只四sgRNA靶向Tyr的小鼠(sgRNA-Tyr-B+C+D+E靶向产生的白化小鼠)的尾巴组织进行鉴定后，本发明人发现，五只小鼠中Tyr的敲除完全是由于整个外显子的敲除所导致的，而剩下的一只小鼠中Tyr的敲除80％是由于外显子敲除而20％是由于插入缺失导致的移码突变(图3A和3B；图8A和图8B)。
考虑到尾部组织未必具有代表性，本发明人又对单sgRNA组(组I)和四sgRNA组(组IV)做了全囊胚检测。正如预计的那样，组IV所有的囊胚(n＝12)表现出了野生型Tyr等位基因的完全缺失，包括74％的外显子大片段删除(＞30bp)以及26％的插入缺失(＜30bp)。相反，组I的囊胚(12个胚胎中的8个是嵌合体)则表现出不同比例的野生型Tyr等位基因(图3C和3D，图8C-图8F)。
为了排除由于PCR的偏差而低估在整个囊胚中野生型Tyr基因含量的可能性，本发明人进一步在单细胞水平上检测了基因编辑的胚胎。本发明人用PCR技术测定了8到16细胞期胚胎的每个卵裂球的Tyr基因，大约有53％的卵裂球能被成功扩增(图图8G和图8H)。对来自3到4个sgRNA靶向组(组IV)的来自21个胚胎的卵裂球的检测发现，每个卵裂球(n＝116)都表现出野生型Tyr基因的缺失，既有插入缺失又有外显子大片段删除，或者二者皆有(图3E到3I)。相对的，组I(只用一个sgRNA)中来自9个胚胎的58个卵裂球当中，只有8个表现出野生型Tyr基因或是Tyr基因单等位基因突变，这意味着67％的胚胎都是嵌合体(图3E到3I)。
本发明人同样也对进行Tyr基因编辑的两细胞胚胎进行了单细胞分析，发现在所有的两细胞卵裂球(n＝46)中C-CRISPR介导的基因编辑早在两细胞阶段就发生了，而且所有胚胎都是双等位基因上的Tyr敲除。相反，用单个sgRNA靶向的胚胎中在两细胞阶段有非常高的嵌合率(62％)(图9A-G)。
实施例4、无嵌合现象的三基因敲除
为了检测向受精卵中注射C-CRISPR能否一次性敲除多个基因，同时靶向Tet1，Tet2和Tet3三个涉及DNA氧化并功能互为冗余的基因(图4A)。应用于制作体内转录模板的引物如表1中相应序列(Tet1-A～C；Tet2-A～C；Tet3-A～C；及sgRNA-R)；sgRNA靶序列如表2中相应序列。sgRNA-Tet1-A、sgRNA-Tet1-B、sgRNA-Tet1-C靶向Tet1的外显子2；sgRNA-Tet2-A、sgRNA-Tet2-B、sgRNA-Tet2-C靶向Tet2的外显子3；sgRNA-Tet3-A、sgRNA-Tet3-B、sgRNA-Tet3-C靶向Tet1的外显子4。
胚胎移植之后，本发明人收集了E7.5的胚胎并通过分析5’羟甲基化胞嘧啶(5hmC)的含量来检测Tet基因的表达，因为5hmC是Tet蛋白氧化5’甲基胞嘧啶(5mC)的主要产物。在靶向Tet基因的胚胎(组I：用六个sgRNA靶向Tet1和Tet2两个基因，Tet1,2-A+B+C；组III：用九个sgRNA靶向Tet1、Tet2和Tet3三个基因，Tet1,2,3-A+B+C)中，大约有一半的胚胎在E7.5时发生了退化(图4B)，这个和之前的报道是一致的。对外胚层组织切片的5hmC和5mC免疫染色显示，野生型胚胎在所有细胞中都有很高水平的5hmC(图4C)，相比之下，用三个sgRNA三重靶向Tet1，Tet2和Tet3基因的胚胎(组II，对每个基因使用一个sgRNA，Tet1，2，3-A)则表现出嵌合状的5hmC染色，某些细胞的5hmC水平很高(图4C)。不仅如此，用六个sgRNA双重靶向Tet1和Tet2的胚胎(每个基因三个sgRNA)也表现出类似的5hmc嵌合染色。然而，用九个sgRNA三重靶向Tet1，Tet2和Tet3基因的胚胎(每个基因三个sgRNA，Tet1，2，3-A)的5hmC则彻底消失了(图4C)，这表明全部三个Tet基因全被删除了。
本发明人用免疫荧光密度测算了5hmC/5mC的平均比值，结果显示在组I和II当中比例减半，而在组III当中则变得更低(图4D)。
用六个sgRNA双重删除Tet基因的胚胎出生率有所下降，而九个sgRNA三重删除Tet基因没有活着出生的(图4E)。
这些结果充分证明，用C-CRISP方法一步删除多个基因的效率。
实施例5、对通过C-CRISPR制作的F0小鼠进行表型分析
本发明人继而采用C-CRISPR方法对多个Y染色体基因的功能做了表型分析，同时也测试了在小鼠中一步法基因敲除用于大量基因快速功能筛选的效率。Y染色体高度特化，专用于雄性的性别分化与生育。然而迄今为止只有少数几个基因被靶向敲除并检测了其生物学功效。所以，本发明人决定分别靶向其中的八个单拷贝Y染色体基因：Zfy1；Ube1y1；Kdm5d；Eif2s3y；Ddx3y；Usp9y；Sry；Erdr1(图5A)。这些基因中Sry和Eif2s3y曾被常规方法敲除过。应用于制作体内转录模板的引物如表1中相应序列(Zfy1-A～B；Ube1y1-A～B；Kdm5d-A～B；Eif2s3y-A～B；Ddx3y-A～B；Usp9y-A～B；Sry-A～B；Erdr1-A～B；及sgRNA-R)；sgRNA靶序列如表2中相应序列。
在八个被检测的基因当中，本发明人发现靶向删除同时存在于X染色体和Y染色体上的Erdr1基因是胚胎致死的。而分别删除其余七个基因则对出生率没有显著影响，而且对这些小鼠的基因型鉴定结果也证实了整个基因被删除(图10)。与之前的研究结果相一致的是，所有敲除Sry基因的小鼠都长有雌性外生殖器和乳头，这表明删除Sry基因会导致小鼠性别逆转。相对的，靶向其它六个基因的小鼠则有正常的性别比例(图5B和5C)。靶向删除Eif2s3y基因的雄性小鼠不能生育而且睾丸发育不良(图5D到5F，表5)，这和之前的一项研究结果一致。有趣的是，删除其它五个基因(Zfy1，Ube1y1，Kdm5d，Ddx3y，Usp9y)的雄性小鼠都是可生育的，可以和野生型雌性小鼠配种产生后代(表5、表6)。然而删除Zfy1或Kdm5d基因的小鼠睾丸体重比较低，而删除Ube1y1或Dxd3y基因的小鼠的活力正常的精子比例更低(图5D到5F，表5)。对睾丸组织的组织学分析也表明六个基因中只有Eif2s3y基因会导致精原细胞分化缺陷(图5G)。这些结果表明，C-CRISPR法可通过建立基因功能完全敲除的F0代小鼠，用于基因功能的快速表型分析。
表5、C-CRISPR导致的Y染色体基因删除的F0和F1小鼠的平均产仔数
Figure PCTCN2018081016-appb-000015
Figure PCTCN2018081016-appb-000016
表6、C-CRISPR导致的Y染色体基因删除的F0和F1小鼠的表型
Figure PCTCN2018081016-appb-000017
Figure PCTCN2018081016-appb-000018
Figure PCTCN2018081016-appb-000019
WT：野生型；Pure KO：完全敲除突变小鼠。
实施例6、一步法得到无嵌合的基因敲除猴
接下来本发明人检测了C-CRISPR方法能否用于构建非嵌合的基因编辑猴。本发明人对食蟹猴(Macaca fascicularis)的Prrt2基因做了靶向敲除，以便构建人类发作性运动诱发性运动障碍(Paroxysmal Kinesigenic Dyskinesia，PKD)的动物模型。
本发明人首先使用传统的CRISPR/Cas9策略，针对Prrt2基因的2号外显子设计了十个sgRNA(指每次单独注射一个sgRNA)，向胚胎中注射(每次注射一种)这些sgRNA，然后在囊胚阶段进行检测。应用于制作体内转录模板的引物如表1中相应序列(Prrt2-A及sgRNA-R)；sgRNA靶序列如表2中相应序列。结果发现其中只有一个sgRNA(sgRNA-Prrt2-A)在靶向基因上比较高效(图6A和图11)。接着，本发明人将sgRNA-Prrt2-A和Cas9 mRNA一齐注入猴子受精卵，并且将55个这样的胚胎在二细胞期移植到了17个代孕母亲体内。得到了6只流产死胎和4只活猴子。对十只猴子的尾部做了基因组分析之后，发现两只死胎(#2和#3)和3只活猴(#4、#8和#10)有20-60％不同程度的Prrt2的删除，而其余5只猴子没有被基因编辑(图6F，表7)。
为了尝试C-CRISPR方法，本发明人又另外构建了靶向Prrt2基因的3号外显子的3个sgRNA(sgPrrt2-B至sgPrrt2-D)，并且在受精卵当中分别注射这些sgRNA后等胚胎发育到囊胚阶段再检测其效率。应用于制作体内转录模板的引物如表1中相应序列(Prrt2-B～D)；sgRNA靶序列如表2中相应序列。本发明人发现所有这些sgRNA都能在靶向位点引入DNA切割(图11A)。然后将所有这些sgRNA(sgRNA-Prrt2-B+C+D)连同Cas9 mRNA一齐注入猴子受精卵当中。等胚胎发育到八细胞期后，本发明人检测了所注胚胎的所有卵裂球，结果发现除了一个卵裂球是单等位基因突变以外，所有别的有PCR信号的卵裂球都有Prrt2基因的双等位基因突变(n＝33，11枚胚胎)，这表明几乎完全没有嵌合性(11个胚胎中有10个完全敲除的胚胎)(图6B到6E，图11B)。相对的，对照组，也就是只注射单个sgRNA(sgPrrt2-C)的八个八细胞胚胎的卵裂球则有90％是野生型，另 有10％为单等位基因突变，与高嵌合性的特点相符合(3个嵌合胚胎与5个野生型胚胎)(图6B到6E；表7)。
在病人中PRRT2的基因发生纯合突变的话会伴随一系列的临床症状，如各类阵发性运动障碍，良性家族性婴儿癫痫，偏瘫性偏头痛，阵发性斜颈，发作性共济失调，甚至发生智力障碍。接下来，检测了Prrt2敲除猴能否也表现出这些症状。本发明人观察到了在运动功能方面的异常类似于阵发性运动障碍：由sgRNA-Prrt2-B+C+D靶向获得存活猴#11出生的时候并没有明显的缺陷，并在刚出生不久的一段时间里表现出正常的活动状态(图6H)。到18天后这只猴子才表现出类似发作性运动诱发性运动障碍的症状，具有四肢的异常动作(躯干弯曲以及偶尔的紧紧握拳)。这种动作在同年龄正常的野生型猴子身上是观察不到的，这些异常动作可能是由突然性的自主运动所诱发的(类似PKD的症状)。在61天大时，猴#11表现出严重的运动障碍，包括肢体震颤以及下肢僵硬导致的猴无法行走和坐下。在105天时，猴#11在接受下肢训练后恢复了行走能力。这些类似PKD的症状消失了，但是在一定的应激之下，比如翻跟斗或是摆尾巴同样也能引发不正常的四肢运动以及手脚握拳(Paroxysmal nonkinesigenic dyskinesia，阵发性运动障碍，PNKD)。本发明人还观察到有诸如在触摸和极端睡意时手脚握拳等异常行为。在5个月大时，猴#11身上还发展出类似头痛的症状，会经常用头撞击笼内的物品或笼子地板上，同时握拳并晃动身体，这种类似头痛的症状持续了大约一个月。猴#11在一岁时还出现了学习障碍，表现为它无法学会用手和吃食物，因此它进食是靠把头低下来凑近地板上的食物，而不是用它的手来拿。许多这些表型都类似于表现人类Prrt2相关异常的症状。
相反，用CRISPR/Cas9系统注射单个sgRNA所获得的#4、#8和#10猴和用C-CRISPR方法获得的#12猴，它们同为嵌合体，在长达1年的观察期中，均未发现有运动障碍。通过对#12猴的成纤维细胞(n＝25)和血细胞(n＝43)的序列分析，发现在46-64％的细胞中有Prrt2点突变(335N变为335A)，其余细胞中为大片段的外显子删除(图6G；图11D至F)。这个点突变可能不足以来破坏Prrt2的功能，所以导致这种无异常行为的表型。对于用单个sgRNA注射所获得流产的嵌合的#3猴，蛋白质印迹分析结果表明，皮层和小脑的Prrt2表达大部分但并非全部都被去除了(图6I)。这些结果表明，完全删除猴子的Prrt2对制作类PKD综合征猴子模型而言是必须的。
为了证明在猴子胚胎中完全敲除的可靠性，本发明人同样使用C-CRISPR方法去靶向芳香烃受体核转运蛋白样蛋白1(Arntl)，这个蛋白是节律钟的一个核心成分。本发明人首先设计了4个sgRNA(sgRNA-Arntl-A,B,G和J)来靶向Arntl的两个有功能的PAS结构域(分别在8号和13号外显子)，然后在猴子胚胎中检测DNA剪切效率(图12A)。
本发明人发现，sgRNA-Arntl-G和sgRNA-Arntl-J在13号外显子上作用效果明显(图 12B)。为了选择更多的sgRNA来靶向Arntl，本发明人针对13号外显子设计了另外7个sgRNA(sgRNA-Arntl-C,D,E,F,H,I和K)(图12A)。通过T7E1分析法在猴子细胞COS-7中检测了sgRNA剪切效率，本发明人发现，同之前在猴子胚胎上测试的两个sgRNA(sgRNA-Arntl-G和J)一样，5个sgRNA(sgRNA-Arntl-D,E,H,I和K)具有高的DNA剪切效率(37％至60％)(图12C和12D)。考虑到每个sgRNA之间的距离，本发明人选择了sgRNA-Arntl-D、sgRNA-Arntl-G还有sgRNA-Arntl-J共同注射到猴子胚胎中，然后在八细胞期的胚胎中进行基因型分析。本发明人发现所有的八个检测的胚胎中都有Arntl基因的完全删除(图12E至12G，表1)。总之，这些结果表明C-CRISPR方法能够在猴子中做到完全的基因敲除。
表7、猴子中C-CRISPR介导的基因编辑
Figure PCTCN2018081016-appb-000020
/，未确定；WT，野生型；-，阴性；+，阳性。
1编辑的Prrt2几乎在所有细胞中包含大片段外显子缺失。
2编辑的Prrt2在约50-80％的细胞中包含大片段外显子缺失，其余仅呈现Prrt2点突变(335N→335A)。
实施例7、C-CRISPR方法在小鼠和猴子内的脱靶效果
本发明人检测C-CRISPR方法的多个sgRNA靶向是否会比单个sgRNA靶向带来更多的脱靶效应。本发明人针对四个sgRNA靶向(Tyr-B+C+D+E-#1,#2,#3,#4,#5,#6)的六只小鼠和三个sgRNA靶向(#11，#12)的两只猴子中所使用的每个sgRNA的多达10个可能会脱靶位点进行了检测。从这些基因组位点扩增出来的PCR产物的DNA测序表明这些位点没有突变发生(图13)。本发明人也对于样品进行了全基因组测序(WGS)，同样在最有可能发生脱靶的20个位点处没有发现脱靶效应。因此，C-CRISPR在基因编辑过的小鼠和猴子中不会引入明显的脱靶改变，这个超出普遍对于CRISPR/Cas9介导编辑的预期。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (31)

  1. 制备基因敲除的动物细胞的方法，其特征在于，所述方法包括：
    (1)根据待敲除的目标基因的核酸序列，制备两条或多条靶向目标基因上不同靶位点的sgRNA；
    (2)将(1)的sgRNA或能形成所述sgRNA的核酸、Cas9mRNA或能形成所述Cas9mRNA的核酸共转入动物细胞中，获得基因敲除的动物细胞。
  2. 如权利要求1所述的方法，其特征在于，所述的动物细胞为动物受精卵，所述的受精卵可发育为目标基因的基因功能完全敲除或大部分敲除的基因敲除动物。
  3. 制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的方法，其特征在于，所述方法包括：
    (1)根据待敲除的目标基因的核酸序列，制备两条或多条靶向目标基因上不同靶位点的sgRNA；
    (2)将(1)的sgRNA或能形成所述sgRNA的核酸、Cas9mRNA或能形成所述Cas9mRNA的核酸共转入受精卵中，获得基因敲除的动物受精卵；
    (3)使(2)的受精卵发育产生所述的目标基因的基因功能完全敲除或大部分敲除的基因敲除动物。
  4. 如权利要求1～3任一所述的方法，其特征在于，所述的两条或多条靶向目标基因上不同靶位点的sgRNA，靶向的目标基因上的靶位点之间间隔为9～500bp；较佳地为10～300bp。
  5. 如权利要求1～3任一所述的方法，其特征在于，采用多条靶向目标基因上不同靶位点的sgRNA；如为3～30条。
  6. 如权利要求5所述的方法，其特征在于，所述的多条靶向目标基因上不同靶位点的sgRNA，其中至少2条所靶向的目标基因上的靶位点位于同一外显子区域。
  7. 如权利要求5所述的方法，其特征在于，所述的多条靶向目标基因上不同靶位点的sgRNA，在所靶向的目标基因上的靶位点区域引入移码突变；和/或引入插入缺失； 和/或引入大片段缺失。
  8. 如权利要求1～3任一所述的方法，其特征在于，所述的动物为哺乳动物，包括：人，非人灵长类动物，鼠，家畜。
  9. 如权利要求1～3任一所述的方法，其特征在于，所述的sgRNA的核酸序列的上游携带启动子，较佳地该启动子为T7启动子，U6启动子；或
    所述的Cas9mRNA的核酸序列的上游携带启动子，较佳地该启动子为T7启动子。
  10. 如权利要求2～3任一所述的方法，其特征在于，所述的目标基因的基因功能大部分敲除的基因敲除动物的体内，待敲除目标基因未发生有效敲除的细胞占总细胞数的20％以下；更佳的15％以下；更佳地10％以下；进一步更佳地5％以下。
  11. 如权利要求1～3任一所述的方法，其特征在于，所述的待敲除的目标基因为GFP基因，所述的sgRNA为2，3或4条，各sgRNA靶向的目标基因上的靶位点之间间隔为30～200bp；较佳地40～150bp；更佳地50～130bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的2，3或4个：SEQ ID NO:48，SEQ ID NO:49，SEQ ID NO:50，SEQ ID NO:51。
  12. 如权利要求1～3任一所述的方法，其特征在于，所述的待敲除的目标基因为Tyr基因，所述的sgRNA为3，4，5或6条，各sgRNA靶向的目标基因的外显子4及任选地与该外显子邻近的内含子，且sgRNA靶向的靶位点之间间隔为10～100bp；较佳地10～70bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的3，4，5或6个：SEQ ID NO:52，SEQ ID NO:53，SEQ ID NO:54，SEQ ID NO:55，SEQ ID NO:56，SEQ ID NO:57；更佳地为选自下组的3或4个：SEQ ID NO:53，SEQ ID NO:54，SEQ ID NO:55，SEQ ID NO:56。
  13. 如权利要求1～3任一所述的方法，其特征在于，所述的待敲除的目标基因为Tet1基因，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子2，且各sgRNA靶向的靶位点之间间隔为80～200bp；较佳地100～190bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:58，SEQ ID  NO:59，SEQ ID NO:60。
  14. 如权利要求1～3任一所述的方法，其特征在于，所述的待敲除的目标基因为Tet2基因，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子3，且各sgRNA靶向的靶位点之间间隔为100～150bp；较佳地110～135bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:61，SEQ ID NO:62，SEQ ID NO:63。
  15. 如权利要求1～3任一所述的方法，其特征在于，所述的待敲除的目标基因为Tet3基因，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子4，且各sgRNA靶向的靶位点之间间隔为180～280bp；较佳地190～270bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:64，SEQ ID NO:65，SEQ ID NO:66。
  16. 如权利要求1～3任一所述的方法，其特征在于，所述的待敲除的目标基因为Prrt2基因，所述的sgRNA为3或4条，至少3条sgRNA靶向的目标基因的外显子3，且外显子3上sgRNA靶向的靶位点之间间隔为10～50bp；较佳地15～40bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:83，SEQ ID NO:84，SEQ ID NO:85，SEQ ID NO:86；更佳地为：SEQ ID NO:84，SEQ ID NO:85，SEQ ID NO:86。
  17. 如权利要求1～3任一所述的方法，其特征在于，所述的待敲除的目标基因为ArntL基因，所述的sgRNA为3，4，5，6，7，8或9条，靶向的目标基因的外显子13，且外显子13上sgRNA靶向的靶位点之间间隔为10～80bp；较佳地40～70bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的3，4，5或6个：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；更佳地为：SEQ ID NO:92，SEQ ID NO:95，SEQ ID NO:98。
  18. 如权利要求1～3任一所述的方法，其特征在于，所述的待敲除的目标基因为Y染色体基因；较佳地为Zfy1；Ube1y1；Kdm5d；Eif2s3y；Ddx3y；Usp9y；Sry；Erdr1；
    更优选地，所述的待敲除的目标基因为Zfy1，所述的sgRNA靶向的目标基因上的 靶位点为SEQ ID NO:67，SEQ ID NO:68；或
    所述的待敲除的目标基因为Ube1y1，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:69，SEQ ID NO:70；或
    所述的待敲除的目标基因为Kdm5d，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:71，SEQ ID NO:72；或
    所述的待敲除的目标基因为Eif2s3y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:73，SEQ ID NO:74；或
    所述的待敲除的目标基因为Ddx3y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:75，SEQ ID NO:76；或
    所述的待敲除的目标基因为Usp9y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:77，SEQ ID NO:78；或
    所述的待敲除的目标基因为Sry，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:79，SEQ ID NO:80；或
    所述的待敲除的目标基因为Erdr1，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:81，SEQ ID NO:82。
  19. 权利要求1～18任一所述的方法的用途，用于制备基因敲除的动物细胞、动物受精卵或动物，其中所述的动物是目标基因的基因功能完全敲除或大部分敲除的动物；所述的目标基因的基因功能大部分敲除的动物的体内，待敲除目标基因未发生有效敲除的细胞占总细胞数的20％以下；更佳的15％以下；更佳地10％以下；进一步更佳地5％以下。
  20. 如权利要求19所述的用途，其特征在于，所述的目标基因的基因功能完全敲除或大部分敲除的动物，为仅经历一个生育周期即获得的动物。
  21. 权利要求1～18任一所述的方法的用途，用于进行动物基因功能研究；或用于制备目标基因的基因功能完全敲除或大部分敲除的动物，并将之用于基因功能研究，或将之用于胚胎发育研究。
  22. 用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为GFP基因，其特征在于，所述的sgRNA为2，3或4条，各sgRNA靶向的目标基因上的靶位点之间 间隔为30～200bp；较佳地40～150bp；更佳地50～130bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的2，3或4个：SEQ ID NO:48，SEQ ID NO:49，SEQ ID NO:50，SEQ ID NO:51。
  23. 用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Tyr基因，其特征在于，所述的sgRNA为3，4，5或6条，各sgRNA靶向的目标基因的外显子4及任选地与该外显子邻近的内含子，且sgRNA靶向的靶位点之间间隔为10～100bp；较佳地10～70bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的3，4，5或6个：SEQ ID NO:52，SEQ ID NO:53，SEQ ID NO:54，SEQ ID NO:55，SEQ ID NO:56，SEQ ID NO:57；更佳地为选自下组的3或4个：SEQ ID NO:53，SEQ ID NO:54，SEQ ID NO:55，SEQ ID NO:56。
  24. 用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Tet1基因，其特征在于，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子2，且各sgRNA靶向的靶位点之间间隔为80～200bp；较佳地100～190bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:58，SEQ ID NO:59，SEQ ID NO:60。
  25. 用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Tet2基因，其特征在于，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子3，且各sgRNA靶向的靶位点之间间隔为100～150bp；较佳地110～135bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:61，SEQ ID NO:62，SEQ ID NO:63。
  26. 用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Tet3基因，其特征在于，所述的sgRNA为3条，各sgRNA靶向的目标基因的外显子4，且各sgRNA靶向的靶位点之间间隔为180～280bp；较佳地190～270bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:64，SEQ ID NO:65，SEQ ID NO:66。
  27. 用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为Prrt2基因，其特征在于，所述的sgRNA为3或4条，至少3条sgRNA靶向的目标基因的外显子3，且外显子3上sgRNA靶向的靶位点之间间隔为10～50bp；较佳地15～40bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为：SEQ ID NO:83，SEQ ID NO:84，SEQ ID NO:85，SEQ ID NO:86；更佳地为：SEQ ID NO:84，SEQ ID NO:85，SEQ ID NO:86。
  28. 用于制备目标基因的基因功能完全敲除或大部分敲除基因敲除动物的sgRNA或能形成所述sgRNA的核酸，该sgRNA用于敲除的目标基因为ArntL基因，其特征在于，所述的sgRNA为3，4，5，6，7，8或9条，靶向的目标基因的外显子13，且外显子13上sgRNA靶向的靶位点之间间隔为10～80bp；较佳地40～70bp；
    更优选地，所述的sgRNA靶向的目标基因上的靶位点为选自下组的3，4，5或6个：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；更佳地为：SEQ ID NO:92，SEQ ID NO:95，SEQ ID NO:98。
  29. 用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的sgRNA或能形成所述sgRNA的核酸，其特征在于，该sgRNA用于敲除的目标基因为Y染色体基因Zfy1，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:67，SEQ ID NO:68；或
    该sgRNA用于敲除的目标基因为Y染色体基因Ube1y1，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:69，SEQ ID NO:70；或
    该sgRNA用于敲除的目标基因为Y染色体基因Kdm5d，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:71，SEQ ID NO:72；或
    该sgRNA用于敲除的目标基因为Y染色体基因Eif2s3y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:73，SEQ ID NO:74；或
    该sgRNA用于敲除的目标基因为Y染色体基因Ddx3y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:75，SEQ ID NO:76；或
    该sgRNA用于敲除的目标基因为Y染色体基因Usp9y，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:77，SEQ ID NO:78；或
    该sgRNA用于敲除的目标基因为Y染色体基因Sry，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:79，SEQ ID NO:80；或
    该sgRNA用于敲除的目标基因为Y染色体基因Erdr1，所述的sgRNA靶向的目标基因上的靶位点为SEQ ID NO:81，SEQ ID NO:82。
  30. 用于制备目标基因的基因功能完全敲除或大部分敲除的基因敲除动物的试剂盒，其特征在于，所述的试剂盒中包含权利要求22～29任一所述的用于制备目标基因的基因功能完全敲除或大部分敲除的动物的sgRNA，或能形成所述sgRNA的核酸。
  31. 如权利要求30所述的试剂盒，其特征在于，所述的试剂盒中还包括：
    Cas9mRNA或能形成所述Cas9mRNA的核酸；或
    使用说明书。
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