WO2022062055A1 - Crispr系统及其在制备多基因联合敲除的重症免疫缺陷克隆猪核供体细胞中的应用 - Google Patents

Crispr系统及其在制备多基因联合敲除的重症免疫缺陷克隆猪核供体细胞中的应用 Download PDF

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WO2022062055A1
WO2022062055A1 PCT/CN2020/124634 CN2020124634W WO2022062055A1 WO 2022062055 A1 WO2022062055 A1 WO 2022062055A1 CN 2020124634 W CN2020124634 W CN 2020124634W WO 2022062055 A1 WO2022062055 A1 WO 2022062055A1
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sgrna
plasmid pkg
dqa
il2rg
ada
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French (fr)
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牛冬
汪滔
马翔
曾为俊
王磊
程锐
赵泽英
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南京启真基因工程有限公司
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    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0387—Animal model for diseases of the immune system
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to a CRISPR system and its application in the preparation of multi-gene combined knockout of severe immunodeficiency cloned porcine nuclear donor cells, in particular to a preparation of four genes combined knockout of ADA gene, DQA gene, DRA gene and IL2RG gene Methods, CRISPR systems, sgRNA combinations, plasmid combinations and kits for the removal of severely immunodeficient pig-derived recombinant cells.
  • SCID Severe combined immunodeficiency
  • primary immunodeficiency disease refers to the simultaneous development and differentiation of T cells, B cells and NK cells due to genetic, developmental or infection factors. , proliferation, metabolism or dysfunction.
  • SCID disease in human infants was first reported by Glanzmann and Riniker in 1950. Globally, the neonatal incidence of SCID is about 1/50,000. The disease has an early age of onset, severe clinical manifestations, and high mortality. Most SCIDs are caused by abnormalities of immune-related genes, and the main inheritance modes of SCID include X-linked recessive inheritance and autosomal recessive inheritance. The disease has certain regional and consanguineous characteristics, and it is more common in male patients due to the characteristics of X-linked recessive inheritance.
  • treatments for SCID mainly include bone marrow or hematopoietic stem cell transplantation and gene therapy. Bone marrow or stem cell transplantation is the best treatment for SCID, but finding a suitable donor for the patient is difficult.
  • SCID caused by autosomal recessive inheritance is further divided into SCID caused by nucleotide metabolism-related enzyme defects and major histocompatibility complex (MHC) molecular defects.
  • MHC major histocompatibility complex
  • Defects in the purine nucleotidase-related gene ADA in nucleotide metabolism-related enzymes lead to a large enrichment of intracellular nucleotide metabolites dATP or dGTP, which are selectively toxic to lymphocytes, thereby causing the function of lymphocytes handicap, impairment, or death, leading to SCID.
  • SCID due to molecular defects of the major histocompatibility complex is caused by MHC abnormalities in the short arm of chromosome 6, which are subdivided into MHC class I and MHC class II defects.
  • Class I is caused by TAP gene defect resulting in the unstable structure of MHC class I molecules; class II is caused by gene defects such as DQA, DRA, resulting in no or low expression of MHC class II molecules.
  • a malfunction of the MHC molecule results in the inability of T cells to recognize antigenic signals, resulting in a loss of their associated immune response, which in turn leads to SCID.
  • SCID caused by X-linked recessive inheritance is the most common type, and its pathogenic mutations are mutations in the IL2RG gene encoding the IL-2R ⁇ chain, resulting in dysfunction of the IL-2R ⁇ chain.
  • the IL-2R ⁇ chain also known as the common ⁇ chain, is a variety of cytokine receptors involved in the regulation of immune cell differentiation, development and maturation, such as IL-2, IL-4 and IL-7. Signal transduction molecules commonly used when transducing signals into immune cells after ligand binding. Therefore, dysfunction of the shared ⁇ chain can lead to abnormal function or development of immune cells, thereby triggering SCID.
  • pig As a large animal, pig has been the main source of meat for humans for a long time. It is easy to breed and raise on a large scale, and it has lower requirements on ethics and animal protection. At the same time, pigs are similar in size and organ function to humans, making them an ideal human being. disease model animals. In addition, when studying the effects of biologically active macromolecules or cell therapy, experiments with heterologous animals will result in immune rejection, making effective animal experiments impossible. The use of severe combined immunodeficiency model animals can avoid the problem of immune rejection between xenogeneic species.
  • the invention relates to a CRISPR system and its application in the preparation of multi-gene combined knockout of severe immunodeficiency cloned porcine nuclear donor cells, in particular to a preparation of four genes combined knockout of ADA gene, DQA gene, DRA gene and IL2RG gene Methods, CRISPR systems, sgRNA combinations, plasmid combinations and kits for the removal of severely immunodeficient pig-derived recombinant cells.
  • the invention provides a method for preparing recombinant cells, comprising the following steps: plasmid pKG-T6gRNA (ADA-g7), plasmid pKG-T6gRNA (DQA-gn2), plasmid pKG-T6gRNA (DRA-g1), plasmid pKG- T6gRNA (IL2RG-g7) and plasmid pKG-GE3 were co-transfected into pig cells to obtain recombinant cells with mutations in ADA gene, DQA gene, DRA gene and IL2RG gene.
  • the pig cells may be pig fibroblasts.
  • the porcine cells can specifically be porcine primary fibroblasts.
  • the pig can be Congjiangxiang pig.
  • the recombinant cells prepared by the method also belong to the protection scope of the present invention.
  • the recombinant cells can be any of the following: the monoclonal cell lines numbered 6, 12, 29, 31, 45, and 52 in Tables 1 to 4.
  • the invention also protects the application of the recombinant cells in the preparation of immunodeficiency animal models.
  • the invention also protects the application of the recombinant cells in the preparation of immunodeficiency animal cell models.
  • a cloned pig is obtained by using the recombinant cell as a donor cell for nuclear transplantation using a somatic cell cloning technology, which is an immunodeficiency animal model.
  • the immunodeficiency animal cell model can be prepared by using the immunodeficiency animal model, that is, isolating the corresponding cells of the immunodeficiency animal model as the immunodeficiency animal cell model.
  • the animal model is a pig model.
  • the animal cell model is a pig cell model.
  • the animal is a pig, specifically Congjiangxiang pig.
  • the present invention also provides a sgRNA combination, consisting of sgRNA ADA-g7 , sgRNA DQA-gn2 , sgRNA DRA-g1 and sgRNA IL2RG-g7 .
  • the present invention also provides a plasmid combination, which is composed of plasmid pKG-T6gRNA (ADA-g7), plasmid pKG-T6gRNA (DQA-gn2), plasmid pKG-T6gRNA (DRA-g1) and plasmid pKG-T6gRNA (IL2RG-g7).
  • a plasmid combination which is composed of plasmid pKG-T6gRNA (ADA-g7), plasmid pKG-T6gRNA (DQA-gn2), plasmid pKG-T6gRNA (DRA-g1) and plasmid pKG-T6gRNA (IL2RG-g7).
  • the present invention also provides a kit comprising the sgRNA combination.
  • the present invention also provides a kit comprising the plasmid combination.
  • the kit also includes plasmid pKG-GE3.
  • the present invention also protects the application of the sgRNA combination in the preparation kit.
  • the present invention also protects the application of the plasmid combination in the preparation kit.
  • the invention also protects the application of the plasmid combination and the plasmid pKG-GE3 in the preparation kit.
  • kits are as follows (a) or (b) or (c): (a) preparing recombinant cells; (b) preparing immunodeficiency animal models; (c) preparing immunodeficiency animal cell models.
  • the recombinant cells are first prepared, and then the recombinant cells are used as donor cells for nuclear transplantation to obtain cloned animals by somatic cell cloning technology, which is an immunodeficiency animal model.
  • the immunodeficient animal cell model can be prepared from the immunodeficient animal model, that is, the corresponding cells of the immunodeficient animal model can be isolated as the immunodeficient animal cell model.
  • the animal may in particular be a pig.
  • the animal model is a pig model.
  • the animal cell model is a pig cell model.
  • the recombinant cells are pig recombinant cells.
  • the transformed recipient cells of the recombinant cells are pig cells.
  • the pig cells may be pig fibroblasts.
  • the porcine cells can specifically be porcine primary fibroblasts.
  • the pig can be Congjiangxiang pig.
  • the present invention also protects the application of any of the above sgRNA combinations or any of the above-mentioned plasmid combinations or any of the above-mentioned kits in the preparation of recombinant cells.
  • the recombinant cells are pig recombinant cells.
  • the transformed recipient cells of the recombinant cells are pig cells.
  • the pig cells may be pig fibroblasts.
  • the porcine cells can specifically be porcine primary fibroblasts.
  • the pig can be Congjiangxiang pig.
  • the present invention also protects the application of any of the above-mentioned sgRNA combinations or any of the above-mentioned plasmid combinations or any of the above-mentioned kits in the preparation of immunodeficiency animal models.
  • the present invention also protects the application of any of the above-mentioned sgRNA combinations or any of the above-mentioned plasmid combinations or any of the above-mentioned kits in the preparation of immunodeficiency animal cell models.
  • the recombinant cells are prepared first, and then the recombinant cells are used as donor cells for nuclear transplantation to obtain cloned animals by somatic cell cloning technology, which is an animal model of immunodeficiency.
  • the immunodeficiency animal cell model can be prepared by using the immunodeficiency animal model, that is, isolating the corresponding cells of the immunodeficiency animal model as the immunodeficiency animal cell model.
  • the animal model is a pig model.
  • the animal cell model is a pig cell model.
  • the animal may in particular be a pig.
  • the recombinant cells are pig recombinant cells.
  • the transformed recipient cells of the recombinant cells are pig cells.
  • the pig cells may be pig fibroblasts.
  • the porcine cells can specifically be porcine primary fibroblasts.
  • the pig can be Congjiangxiang pig.
  • Any one of the above recombinant cells is a cell deficient in ADA gene, DQA gene, DRA gene and IL2RG gene.
  • any one of the above-mentioned recombinant cells is a recombinant cell in which ADA gene, DQA gene, DRA gene and IL2RG gene are all mutated.
  • the mutation may be a heterozygous mutation (the corresponding genotype is a heterozygous mutant) or a homozygous mutation (the corresponding genotype is a biallelic same mutant or a biallelic different mutant).
  • sgRNA ADA-g7 target 5'-GGAGGGCGTGGTGTACGTGG-3'.
  • sgRNA DQA-gn2 target 5'-GTAGACATTTAAGCCATAGG-3'.
  • gRNA DRA-g1 target 5'-TCCACGTGGATATGGAAAAG-3'.
  • sgRNA IL2RG-g7 target 5'-TCCCTTCAGAGAATAGATAG-3'.
  • the plasmid pKG-T6gRNA(ADA-g7) was transcribed to obtain sgRNA ADA-g7 .
  • the plasmid pKG-T6gRNA(DQA-gn2) was transcribed to obtain sgRNA DQA-gn2 .
  • the plasmid pKG-T6gRNA(DRA-g1) was transcribed to obtain sgRNA DRA-g1 .
  • the plasmid pKG-T6gRNA(IL2RG-g7) was transcribed to obtain sgRNA IL2RG-g7 .
  • the target sequence binding region of the sgRNA ADA-g7 is shown in nucleotides 1-20 in SEQ ID NO: 11.
  • the target sequence binding region of the sgRNA DQA-gn2 is shown in nucleotides 1-20 in SEQ ID NO: 21.
  • the target sequence binding region of the sgRNA DRA-gl is shown in nucleotides 1-20 in SEQ ID NO: 28.
  • the target sequence binding region of the sgRNA IL2RG-g7 is shown in nucleotides 1-20 in SEQ ID NO:40.
  • the sgRNA ADA-g7 is shown in SEQ ID NO:11.
  • the sgRNA DQA-gn2 is shown in SEQ ID NO:21.
  • the sgRNA DRA-gl is shown in SEQ ID NO:28.
  • the sgRNA IL2RG-g7 is shown in SEQ ID NO:40.
  • the plasmid pKG-T6gRNA (ADA-g7) is obtained by inserting the coding sequence of the target sequence binding region of sgRNA ADA-g7 into the pKG-U6gRNA vector by means of restriction endonuclease BbsI.
  • the plasmid pKG-T6 gRNA (DQA-gn2) is obtained by inserting the coding sequence of the target sequence binding region of sgRNA DQA-gn2 into the pKG-U6 gRNA vector by means of restriction endonuclease BbsI.
  • the plasmid pKG-T6gRNA (DRA-g1) was obtained by inserting the coding sequence of the target sequence binding region of sgRNA DRA-g1 into the pKG-U6gRNA vector by means of the restriction endonuclease BbsI.
  • the plasmid pKG-T6gRNA (IL2RG-g7) was obtained by inserting the coding sequence of the target sequence binding region of sgRNA IL2RG-g7 into the pKG-U6gRNA vector by means of restriction endonuclease BbsI.
  • the specific fusion gene encodes a specific fusion protein
  • the specific fusion protein sequentially includes the following elements from the N-terminus to the C-terminus: two nuclear localization signals (NLS), Cas9 protein, two nuclear localization signals, self-cleaving polypeptide P2A, fluorescent reporter protein, self-cleaving polypeptide T2A, anti- sexually selectable marker protein;
  • the expression of the specific fusion gene is promoted by the EF1a promoter
  • the specific fusion gene downstream has a WPRE sequence element, a 3'LTR sequence element and a bGH poly(A) signal sequence element.
  • the plasmid pKG-GE3 has the following elements in sequence: CMV enhancer, EF1a promoter, the specific fusion gene, WPRE sequence element, 3'LTR sequence element, and bGH poly(A) signal sequence element.
  • the two nuclear localization signals upstream of Cas9 protein are SV40 nuclear localization signals
  • the two nuclear localization signals downstream of Cas9 protein are nucleoplasmin nuclear localization signals.
  • the fluorescent reporter protein may specifically be an EGFP protein.
  • the resistance selection marker protein may specifically be a Puromycin protein.
  • the amino acid sequence of the self-cleaving polypeptide P2A is "ATNFSLLKQAGDVEENPGP" (the position of self-cleavage is between the first amino acid residue and the second amino acid residue from the C-terminus).
  • the amino acid sequence of the self-cleaving polypeptide T2A is "EGRGSLLTCGDVEENPGP" (the cleavage position where self-cleavage occurs is between the first amino acid residue and the second amino acid residue from the C-terminus).
  • the specific fusion gene is specifically shown in nucleotides 911-6706 in SEQ ID NO: 2.
  • the CMV enhancer is shown as nucleotides 395-680 in SEQ ID NO:2.
  • the EF1a promoter is shown at nucleotides 682-890 in SEQ ID NO:2.
  • the WPRE sequence elements are shown at nucleotides 6722-7310 in SEQ ID NO:2.
  • the 3' LTR sequence elements are shown in SEQ ID NO:2 at nucleotides 7382-7615.
  • the bGH poly(A)signal sequence element is shown at nucleotides 7647-7871 in SEQ ID NO:2.
  • Plasmid pKG-GE3 is specifically shown in SEQ ID NO: 2.
  • Plasmid pKG-U6gRNA is specifically shown as SEQ ID NO:3.
  • Pig ADA gene information encoding adenosine deaminase; located on chromosome 17; GeneID is 100625920, Sus scrofa.
  • the protein encoded by the porcine ADA gene is shown in SEQ ID NO:4.
  • the porcine ADA gene has 12 exons, of which the 4th exon and its upstream and downstream sequences of 500 bp are shown in SEQ ID NO: 5.
  • the porcine ADA gene is a gene with a target of sgRNA ADA-g7 .
  • the porcine ADA gene is a gene having the segment shown in SEQ ID NO:5.
  • Pig DQA gene information encoding SLA class II histocompatibility antigen, DQ haplotype D alpha chain precurso; located on chromosome 7; GeneID is 100153387, Sus scrofa.
  • the protein encoded by the porcine DQA gene is shown in SEQ ID NO: 14 (exemplary sequence in NCBI).
  • the porcine DQA gene has 5 exons, of which the second exon and its upstream and downstream sequences of 500 bp are shown in SEQ ID NO: 15 (sequencing results of Jiangxiang pigs in the present invention).
  • the porcine DQA gene is a gene with a target of sgRNA DQA-gn2 .
  • the porcine DQA gene is a gene having the segment shown in SEQ ID NO:15.
  • Pig DRA gene information encoding MHC class II DR-alpha precursor; located on chromosome 7; GeneID is 100135040, Sus scrofa.
  • the protein encoded by the porcine DRA gene is shown in SEQ ID NO: 26 (exemplary sequence in NCBI).
  • the pig DRA gene has 5 exons, of which the second exon and its upstream and downstream sequences of 500 bp are shown in SEQ ID NO: 27 (sequencing results of Jiangxiang pigs in the present invention).
  • the porcine DRA gene is a gene with a target of sgRNA DRA-gl .
  • the porcine DRA gene is a gene having the segment shown in SEQ ID NO:27.
  • Pig IL2RG gene information encoding interleukin 2 receptor subunit gamma; located on the X chromosome; GeneID is 397156, Sus scrofa.
  • the protein encoded by the porcine IL2RG gene is shown in SEQ ID NO:32.
  • the porcine IL2RG gene has 9 exons, of which the 4th exon and its upstream and downstream sequences of 500 bp are shown in SEQ ID NO: 33.
  • the porcine IL2RG gene is a gene with a target of sgRNA IL2RG-g7 .
  • the porcine DRA gene is a gene having the segment shown in SEQ ID NO:33.
  • any of the above-mentioned immunodeficiency may specifically be a severe immunodeficiency.
  • the present invention at least has the following beneficial effects:
  • the research object (pig) of the present invention has better applicability than other animals (mice, primates). So far, no large animal model of severe immunodeficiency has been successfully developed. Rodents such as rats and mice are very different from humans in terms of body size, organ size, physiology, and pathology, and cannot truly simulate the normal physiological and pathological states of humans. Studies have shown that more than 95% of drugs proven to be effective in rats and mice are ineffective in human clinical trials. As far as large animals are concerned, primates are the closest relatives to humans, but they are small in size, late in sexual maturity (mating at the age of 6-7), and are singleton animals. Also high. In addition, primate cloning is inefficient, difficult and costly.
  • Pig does not have the above shortcomings.
  • Pig is the closest animal to humans except primates. Its body shape, body weight, organ size, etc. are similar to those of humans. Aspects are very similar to humans.
  • pigs have early sexual maturity (4-6 months), high fecundity, multiple litters, and can form a larger group within 2-3 years.
  • the cloning technology of pigs is very mature, and the cost of cloning and feeding is much lower than that of primates; and pigs, as human carnivores for a long time, use pigs as disease model animals in terms of animal protection and ethics. The resistance is relatively small .
  • the genotype of the obtained cells can be analyzed through the sequencing result of the PCR product of the target gene (homozygous mutations include biallelic identical mutations and biallelic different mutations) , heterozygous mutation or wild type), the probability of obtaining homozygous mutation is 10% to 20%; in addition, using the obtained homozygous mutant monoclonal cell line for somatic cell nuclear transfer can directly obtain the clone containing the homozygous mutation of the target gene pigs, and this homozygous mutation can be stably inherited.
  • Figure 1 is a schematic diagram of the structure of plasmid pX330.
  • Figure 2 is a schematic diagram of the structure of plasmid pKG-GE3.
  • Figure 3 is a schematic diagram of the structure of plasmid pKG-U6 gRNA.
  • Figure 4 is a schematic diagram of inserting a DNA molecule of about 20 bp (for transcription to form a target sequence binding region of the gRNA) into the plasmid pKG-U6 gRNA.
  • FIG. 5 is an electropherogram of three groups of MSTN in step 3 of Example 2.
  • FIG. 6 is the electropherogram of three groups of FNDC5 in step 3 of Example 2.
  • FIG. 6 is the electropherogram of three groups of FNDC5 in step 3 of Example 2.
  • FIG. 7 is an electrophoresis image after PCR amplification is performed using the genomic DNA of 8 pigs as a template and a primer pair consisting of ADA-GT-F259/ADA-GT-R1005 in step 1 of Example 3.
  • FIG. 7 is an electrophoresis image after PCR amplification is performed using the genomic DNA of 8 pigs as a template and a primer pair consisting of ADA-GT-F259/ADA-GT-R1005 in step 1 of Example 3.
  • FIG. 7 is an electrophoresis image after PCR amplification is performed using the genomic DNA of 8 pigs as a template and a primer pair consisting of ADA-GT-F259/ADA-GT-R1005 in step 1 of Example 3.
  • FIG. 7 is an electrophoresis image after PCR amplification is performed using the genomic DNA of 8 pigs as a template and a primer pair consisting of ADA-GT-F259/ADA-GT-R1005 in step 1 of Example 3.
  • FIG. 8 shows various double-stranded DNA molecules with sticky ends in step 3 of Example 3.
  • FIG. 9 is a graph of sequencing peaks in step 4 of Example 3.
  • FIG. 9 is a graph of sequencing peaks in step 4 of Example 3.
  • FIG. 10 is an electropherogram after PCR amplification is performed using the genomic DNA of 8 pigs as a template and a primer pair consisting of DQA-GT-F534/DQA-GT-R1332 in step 1 of Example 4.
  • FIG. 10 is an electropherogram after PCR amplification is performed using the genomic DNA of 8 pigs as a template and a primer pair consisting of DQA-GT-F534/DQA-GT-R1332 in step 1 of Example 4.
  • FIG. 11 shows various double-stranded DNA molecules with sticky ends in step 3 of Example 4.
  • FIG. 12 is a graph of sequencing peaks in step 4 of Example 4.
  • FIG. 12 is a graph of sequencing peaks in step 4 of Example 4.
  • FIG. 13 is an electropherogram after PCR amplification is performed using the genomic DNA of 8 pigs as a template and a primer pair composed of DRA-GT-F326/DRA-GT-R1192 in step 1 of Example 5.
  • FIG. 13 is an electropherogram after PCR amplification is performed using the genomic DNA of 8 pigs as a template and a primer pair composed of DRA-GT-F326/DRA-GT-R1192 in step 1 of Example 5.
  • FIG. 14 shows various double-stranded DNA molecules with sticky ends in step 3 of Example 5.
  • FIG. 15 is a graph of sequencing peaks in step 4 of Example 5.
  • FIG. 16 is an electrophoresis image after PCR amplification is performed using the genomic DNA of 8 pigs as a template and a primer pair consisting of IL2RG-GT-F4543/IL2RG-GT-R5180 in step 1 of Example 6.
  • FIG. 16 is an electrophoresis image after PCR amplification is performed using the genomic DNA of 8 pigs as a template and a primer pair consisting of IL2RG-GT-F4543/IL2RG-GT-R5180 in step 1 of Example 6.
  • FIG. 17 shows various double-stranded DNA molecules with sticky ends in step 3 of Example 6.
  • FIG. 18 is a graph of sequencing peaks in step 4 of Example 6.
  • FIG. 19 is an electropherogram of the target gene PCR product of the monoclonal cells obtained in Example 7 (a primer pair consisting of ADA-nnF229 and ADA-nnR456 is used).
  • FIG. 20 is an electropherogram of the target gene PCR product of the monoclonal cells obtained in Example 7 (a primer pair composed of DQA-F643 and DQA-R1022 is used).
  • FIG. 21 is an electrophoresis image of the target gene PCR product of the monoclonal cells obtained in Example 7 (a primer pair composed of DRA-F573 and DRA-R968 was used).
  • FIG. 22 is an electrophoresis image of the target gene PCR product of the monoclonal cells obtained in Example 7 (a primer pair composed of IL2RG-nF33 and IL2RG-nR460 was used).
  • FIG. 23 is the sequence peak diagram of the target genes of some monoclonal cells in Table 1.
  • FIG. 24 is a graph of the target gene sequencing peaks of some of the monoclonal cells in Table 2.
  • FIG. 25 is a graph of the target gene sequencing peaks of some of the monoclonal cells in Table 3.
  • FIG. 26 is a graph of the target gene sequencing peaks of some of the monoclonal cells in Table 4.
  • the 8 pigs in Examples 3 to 6 are all newly born Congjiangxiang pigs, including 4 females (named 1, 2, 3, 4 respectively) and 4 males (named A, B, C, D respectively) .
  • the method of preparing pig primary fibroblasts 1Take 0.5g of pig ear tissue, remove the hair, soak it in 75% alcohol for 30-40s, and then wash it with PBS buffer containing 5% (volume ratio) Penicillin-Streptomycin (Gibco).
  • porcine primary fibroblasts used in Examples 2 to 7 were obtained from the pig designated 2 above (female, blood group AO).
  • Plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 was prepared as shown in SEQ ID NO:1. Plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9, referred to as plasmid pX330.
  • Plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO was prepared, as shown in SEQ ID NO:2. Plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, referred to as plasmid pKG-GE3.
  • Plasmid pKG-U6 gRNA was prepared as shown in SEQ ID NO:3.
  • Plasmid pX330, plasmid pKG-GE3 and plasmid pKG-U6 gRNA are circular plasmids.
  • FIG. 1 A schematic diagram of the structure of plasmid pX330 is shown in Figure 1.
  • the 440-725 nucleotides constitute the CMV enhancer
  • the 727-1208 nucleotides constitute the chicken ⁇ -actin promoter
  • the 1304-1324 nucleotides encode the SV40 nuclear localization signal (NLS).
  • the 1325-5449 nucleotides encode the Cas9 protein
  • the 5450-5497 nucleotides encode the nucleoplasmin nuclear localization signal (NLS).
  • nucleotides 395-680 constitute a CMV enhancer
  • nucleotides 682-890 constitute an EF1a promoter
  • nucleotides 986-1006 encode a nuclear localization signal (NLS)
  • Nucleotides 1016-1036 encode the nuclear localization signal (NLS)
  • nucleotides 1037-5161 encode the Cas9 protein
  • nucleotides 5162-5209 encode the nuclear localization signal (NLS)
  • the 5276-5332 nucleotides encode the self-cleaving polypeptide P2A (the amino acid sequence of the self-cleaving polypeptide P2A is "ATNFSLLKQAGDVEENPGP", and the position of the self-cleaving breakage is the first from the C-terminus.
  • the 5333-6046 nucleotides encode the EGFP protein
  • the 6056-6109 nucleotides encode the self-cleaving polypeptide T2A
  • the amino acid sequence of the self-cleaving polypeptide T2A is " EGRGSLLTCGDVEENPGP"
  • nucleotides 6110-6703 encode Puromycin protein (referred to as Puro protein)
  • Puro protein Puro protein
  • the 7310 nucleotides constitute the WPRE sequence element
  • the 7382-7615 nucleotides constitute the 3'LTR sequence element
  • the 7647-7871 nucleotides constitute the bGH poly(A) signal sequence element.
  • Nos. 911-6706 form a fusion gene and express a fusion protein. Due to the existence of the self-cleaving polypeptide P2A and the self-cleaving polypeptide T2A, the fusion protein spontaneously formed the following three proteins: a protein with Cas9 protein, a protein with EGFP protein, and a protein with Puro protein.
  • the plasmid pKG-GE3 is mainly modified as follows: 1Remove the residual gRNA backbone sequence (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTT) to reduce interference; 2The original chicken ⁇ -actin promoter is transformed into the EF1a promoter with higher expression activity , increase the protein expression ability of Cas9 gene; 3Increase the nuclear localization signal encoding gene (NLS) both upstream and downstream of Cas9 gene to increase the nuclear localization ability of Cas9 protein; 4The original plasmid does not have any eukaryotic cell selection marker, which is not conducive to positive Screening and enrichment of transformed cells, inserting the P2A-EGFP-T2A-PURO coding gene downstream of the Cas9 gene in turn, giving the vector fluorescence and eukaryotic cell resistance screening ability; 5 Inserting WPRE elements and 3'LTR sequence elements to enhance Cas9 The protein translation ability of the
  • the schematic diagram of the structure of plasmid pKG-U6gRNA is shown in Figure 3.
  • the 2280-2539 nucleotides constitute the hU6 promoter, and the 2558-2637 nucleotides are used for transcription to form the gRNA backbone.
  • a DNA molecule of about 20 bp (the target sequence binding region used to transcribe and form gRNA) is inserted into the plasmid pKG-U6gRNA to form a recombinant plasmid.
  • the schematic diagram is shown in Figure 4.
  • the recombinant plasmid is transcribed in the cell to obtain gRNA.
  • Target of MSTN-gRNA1 5'-GCTGATTGTTGCTGGTCCCG-3';
  • Target of MSTN-gRNA2 5'-TTTCCAGGCGAAGTTTACTG-3'.
  • Two gRNA targets located in the FNDC5 gene were selected:
  • FNDC5-gRNA1 target 5'-TGTACTCAGTGTCCTCCTCC-3';
  • Target of FNDC5-gRNA2 5'-GCTCTTCAAGACGCCTCGCG-3'.
  • the primers used to amplify the fragment containing the target were:
  • MSTN-F896 5'-TCTCTCAGACAGTGCAGGCATTA-3';
  • MSTN-R1351 5'-CGTTTCCGTCGTAGCGTGATAAT-3'.
  • FNDC5-F209 5'-CAGTTCTCACTTGATGGCCTTGG-3';
  • FNDC5-R718 5'-AGGGGTCTGGGGAGGAATGG-3'.
  • the plasmid pKG-U6gRNA was taken, digested with the restriction enzyme BbsI, and the vector backbone (a linear large fragment of about 3 kb) was recovered.
  • MSTN-1S and MSTN-1A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends.
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (MSTN-1).
  • MSTN-2S and MSTN-2A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends.
  • the double-stranded DNA molecule with cohesive ends was ligated with the vector backbone to obtain plasmid pKG-U6gRNA (MSTN-2).
  • FNDC5-1S and FNDC5-1A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends.
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (FNDC5-1).
  • FNDC5-2S and FNDC5-2A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends.
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (FNDC5-2).
  • MSTN-1S 5'-caccGCTGATTGTTGCTGGTCCCG-3';
  • MSTN-1A 5'-aaacCGGGACCAGCAACAATCAGC-3'.
  • MSTN-2S 5'-caccgTTTCCAGGCGAAGTTTACTG-3';
  • MSTN-2A 5'-aaacCAGTAAACTTCGCCTGGAAAc-3'.
  • FNDC5-1S 5'-caccgTGTACTCAGTGTCCTCCTCC-3';
  • FNDC5-1A 5'-aaacGGAGGAGGACACTGAGTACAc-3'.
  • FNDC5-2S 5'-caccGCTCTTCAAGACGCCTCGCG-3';
  • FNDC5-2A 5'-aaacCGCGAGGCGTCTTGAAGAGC-3'.
  • MSTN-B group Plasmid pKG-U6gRNA (MSTN-1) and plasmid pKG-U6gRNA (MSTN-2) were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (MSTN-1): 0.46 ⁇ g plasmid pKG-U6gRNA (MSTN-2).
  • MSTN-330 group Plasmid pKG-U6 gRNA (MSTN-1), plasmid pKG-U6 gRNA (MSTN-2) and plasmid pX330 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (MSTN-1): 0.46 ⁇ g plasmid pKG-U6 gRNA (MSTN-2): 1.08 ⁇ g plasmid pX330.
  • MSTN-KG group Plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pKG-GE3 were co-transfected into primary porcine fibroblasts. Ratio: about 200,000 porcine primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (MSTN-1): plasmid 0.46 ⁇ g pKG-U6gRNA (MSTN-2): 1.08 ⁇ g plasmid pKG-GE3.
  • FNDC5-B group Plasmid pKG-U6 gRNA (FNDC5-1) and plasmid pKG-U6 gRNA (FNDC5-2) were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.46 ⁇ g plasmid pKG-U6 gRNA (FNDC5-1): 0.46 ⁇ g plasmid pKG-U6 gRNA (FNDC5-2).
  • FNDC5-330 group Plasmid pKG-U6 gRNA (FNDC5-1), plasmid pKG-U6 gRNA (FNDC5-2) and plasmid pX330 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 porcine primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6 gRNA (FNDC5-1): 0.46 ⁇ g plasmid pKG-U6 gRNA (FNDC5-2): 1.08 ⁇ g plasmid pX330.
  • FNDC5-KG group Plasmid pKG-U6 gRNA (FNDC5-1), plasmid pKG-U6 gRNA (FNDC5-2) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.46 ⁇ g plasmid pKG-U6 gRNA (FNDC5-1): 0.46 ⁇ g plasmid pKG-U6 gRNA (FNDC5-2): 1.08 ⁇ g plasmid pKG-GE3.
  • Co-transfection was performed by electroporation, using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use the complete culture medium to culture for 16-18 hours, and then replace with a new complete culture medium for culture. The total incubation time was 48 hours.
  • step 2 trypsinize and collect cells, extract genomic DNA, perform PCR amplification with primer pairs consisting of MSTN-F896 and MSTN-R1351 (three sets of MSTN), or use FNDC5-F209 and FNDC5- A primer pair consisting of R718 (three sets of FNDC5) was subjected to PCR amplification, followed by electrophoresis.
  • the gene deletion mutation efficiency of MSTN-330 group was 27.6%, and the gene deletion mutation efficiency of MSTN-KG group was 86.5%.
  • the gene deletion mutation efficiency of FNDC5-330 group was 18.6%, and the gene deletion mutation efficiency of FNDC5-KG group was 81.7%.
  • the results showed that compared with the use of plasmid pX330, the use of plasmid pKG-GE3 significantly improved the gene editing efficiency.
  • Pig ADA gene information encoding adenosine deaminase; located on chromosome 17; GeneID is 100625920, Sus scrofa.
  • the protein encoded by the porcine ADA gene is shown in SEQ ID NO:4.
  • the porcine ADA gene has 12 exons, of which the 4th exon and its upstream and downstream sequences of 500 bp are shown in SEQ ID NO: 5.
  • the genomic DNAs of 8 pigs were used as templates, and the primer pair composed of primers ADA-GT-F259/ADA-GT-R1005 was used for PCR amplification, and then electrophoresis was performed, as shown in FIG. 7 .
  • the PCR amplification products were recovered and sequenced, and the sequencing results were compared and analyzed with the ADA gene sequences in the public database. Based on the results of the alignment, primers for detecting mutations (primers themselves avoid possible mutation sites) are designed.
  • the primers designed to detect mutations are: ADA-nnF229/ADA-nnR456.
  • ADA-GT-F259 5'-GTTAAGGATCTGGTGTTGCGGGTG-3';
  • ADA-GT-R1005 5'-GTTCACACTCCTAGACTCCAGCC-3'.
  • ADA-nnF229 5'-GAGGCCGTCAAAAGGATTGC-3';
  • ADA-nnR456 5'-CAAAGTCTCTCTTGGGTCAGGG-3'.
  • the 8 targets are as follows:
  • sgRNA ADA-g1 target 5'-AAGGATTGCCTACGAGTTTG-3';
  • sgRNA ADA-g2 target 5'-TTGGAGTTGGCCAGCAGGTG-3';
  • sgRNA ADA-g3 target 5'-TTTCATCTCCACAAACTCGT-3';
  • sgRNA ADA-g4 target 5'-TCAGCCTGGTTCCAGGGGAT-3';
  • sgRNA ADA-g6 target 5'-CCTGCTGGCCAACTCCAAAG-3';
  • sgRNA ADA-g7 target 5'-GGAGGGCGTGGTGTACGTGG-3';
  • sgRNA ADA-g8 target 5'-CAAGGAGGGCGTGGTGTACG-3';
  • sgRNA ADA-g9 target 5'-TGTGGAGATGAAAGCCAAGG-3'.
  • the plasmid pKG-U6gRNA was taken, digested with the restriction enzyme BbsI, and the vector backbone (a linear large fragment of about 3 kb) was recovered.
  • ADA-g1S and ADA-g1A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 8A).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (ADA-g1).
  • the plasmid pKG-U6gRNA(ADA-g1)) expresses the sgRNA ADA-g1 shown in SEQ ID NO:6.
  • ADA-g2S and ADA-g2A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (Fig. 8B).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (ADA-g2).
  • the plasmid pKG-U6 gRNA (ADA-g2) expresses the sgRNA ADA-g2 shown in SEQ ID NO:7.
  • ADA-g3S and ADA-g3A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (Fig. 8C).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (ADA-g3).
  • the plasmid pKG-U6 gRNA (ADA-g3) expresses the sgRNA ADA-g3 shown in SEQ ID NO:8.
  • ADA-g4S and ADA-g4A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (Fig. 8D).
  • the double-stranded DNA molecule with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (ADA-g4).
  • the plasmid pKG-U6 gRNA (ADA-g4) expresses the sgRNA ADA-g4 shown in SEQ ID NO:9.
  • ADA-g6S and ADA-g6A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (Fig. 8E).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (ADA-g6).
  • the plasmid pKG-U6 gRNA (ADA-g6) expresses the sgRNA ADA-g6 shown in SEQ ID NO:10.
  • ADA-g7S and ADA-g7A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 8F).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (ADA-g7).
  • the plasmid pKG-U6 gRNA (ADA-g7) expresses the sgRNA ADA-g7 shown in SEQ ID NO:11.
  • ADA-g8S and ADA-g8A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 8G).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (ADA-g8).
  • the plasmid pKG-U6 gRNA (ADA-g8) expresses the sgRNA ADA-g8 shown in SEQ ID NO:12.
  • ADA-g9S and ADA-g9A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (Fig. 8H).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (ADA-g9).
  • the plasmid pKG-U6 gRNA (ADA-g9) expresses the sgRNA ADA-g9 shown in SEQ ID NO:13.
  • sgRNA-ADA-1S 5'-caccgAAGGATTGCCTACGAGTTTG-3';
  • sgRNA-ADA-1A 5'-aaacCAAACTCGTAGGCAATCCTTc-3'.
  • sgRNA-ADA-2S 5'-caccgTTGGAGTTGGCCAGCAGGTG-3';
  • sgRNA-ADA-2A 5'-aaacCACCTGCTGGCCAACTCCAAc-3'.
  • sgRNA-ADA-3S 5'-caccgTTTCATCTCCACAAACTCGT-3';
  • sgRNA-ADA-3A 5'-aaacACGAGTTTGTGGAGATGAAAc-3'.
  • sgRNA-ADA-4S 5'-caccgTCAGCCTGGTTCCAGGGGAT-3';
  • sgRNA-ADA-4A 5'-aaacATCCCCTGGAACCAGGCTGAc-3'.
  • sgRNA-ADA-6S 5'-caccgCCTGCTGGCCAACTCCAAAG-3';
  • sgRNA-ADA-6A 5'-aaacCTTTGGAGTTGGCCAGCAGGc-3'.
  • sgRNA-ADA-7S 5'-caccGGAGGGCGTGGTGTACGTGG-3';
  • sgRNA-ADA-7A 5'-aaacCCACGTACACCACGCCCTCC-3'.
  • sgRNA-ADA-8S 5'-caccgCAAGGAGGGCGTGGTGTACG-3';
  • sgRNA-ADA-8A 5'-aaacCGTACACCACGCCCTCCTTGc-3'.
  • sgRNA-ADA-9S 5'-caccgTGTGGAGATGAAAGCCAAGG-3';
  • sgRNA-ADA-9A 5'-aaacCCTTGGCTTTCATCTCCACAc-3'.
  • sgRNA-ADA-1S sgRNA-ADA-1A
  • sgRNA-ADA-2S sgRNA-ADA-2A
  • sgRNA-ADA-3S sgRNA-ADA-3A
  • sgRNA-ADA-4S sgRNA-ADA-4A
  • sgRNA-ADA-6S sgRNA-ADA-6A
  • sgRNA-ADA-7S sgRNA-ADA-7A
  • sgRNA-ADA-8S sgRNA-ADA-8A
  • sgRNA-ADA-9S sgRNA-ADA-9A
  • sgRNA-ADA-9A are single-stranded DNA molecular.
  • Group 1 Plasmid pKG-U6 gRNA (ADA-g1) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (ADA-g1): 1.238 ⁇ g plasmid pKG-GE3.
  • the second group Co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (ADA-g2) and plasmid pKG-GE3. Ratio: about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6 gRNA (ADA-g2): 1.238 ⁇ g plasmid pKG-GE3.
  • the third group the plasmid pKG-U6gRNA (ADA-g3) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (ADA-g3): 1.238 ⁇ g plasmid pKG-GE3.
  • the fourth group co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (ADA-g4) and plasmid pKG-GE3.
  • Ratio about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6 gRNA (ADA-g4): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 5 Co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (ADA-g6) and plasmid pKG-GE3. Ratio: about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6 gRNA (ADA-g6): 1.238 ⁇ g plasmid pKG-GE3.
  • the sixth group co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (ADA-g7) and plasmid pKG-GE3.
  • Ratio about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6 gRNA (ADA-g7): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 7 Co-transfection of plasmid pKG-U6 gRNA (ADA-g8) and plasmid pKG-GE3 into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6 gRNA (ADA-g8): 1.238 ⁇ g plasmid pKG-GE3.
  • the eighth group Co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (ADA-g9) and plasmid pKG-GE3. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6 gRNA (ADA-g9): 1.238 ⁇ g plasmid pKG-GE3.
  • the ninth group porcine primary fibroblasts without any transfection.
  • Co-transfection was performed by electroporation using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use the complete culture medium to culture for 16-18 hours, and then replace with a new complete culture medium for culture. The total incubation time was 48 hours.
  • step 2 the cells were digested with trypsin and collected, the genomic DNA was extracted, PCR amplification was performed using a primer pair composed of ADA-nnF229 and ADA-nnR456, and then electrophoresis and sequencing were performed. The results are shown in Figure 9.
  • the editing efficiency of different targets was obtained by analyzing the sequencing peak map using the Synthego ICE tool.
  • the editing efficiencies of the first to ninth groups of different targets were 19%, 17%, 9%, 0%, 2%, 35%, 29%, 20% and 0%.
  • the results showed that the sixth group had the highest editing efficiency, and sgRNA ADA-g7 was the optimal target.
  • Pig DQA gene information encoding SLA class II histocompatibility antigen, DQ haplotype D alpha chain precurso; located on chromosome 7; GeneID is 100153387, Sus scrofa.
  • the protein encoded by the porcine DQA gene is shown in SEQ ID NO: 14 (exemplary sequence in NCBI).
  • the pig DQA gene has 5 exons, wherein the second exon and its upstream and downstream sequences of 500 bp are shown in SEQ ID NO: 15 (the sequencing result of Congjiangxiang pig in the present invention).
  • the genomic DNAs of 8 pigs were used as templates respectively, and a primer pair consisting of primers DQA-GT-F534/DQA-GT-R1332 was used for PCR amplification, and then electrophoresis was performed, as shown in Figure 10 .
  • the PCR amplification products were recovered and sequenced, and the sequencing results were compared with the DQA gene sequences in the public database. Based on the alignment results, primers for detecting mutations (primers themselves avoid possible mutation sites) are designed.
  • the primers designed to detect mutations are: DQA-F643/DQA-R1022.
  • DQA-GT-F534 5'-TTGCAAAGATAAGGAGGCTTCGC-3';
  • DQA-GT-R1332 5'-AGCTCTTGTTTCCCTTCTGCTCA-3.
  • DQA-F643 5'-CAGATGAAGCCCTTGATATTTGA-3';
  • DQA-R1022 5'-AGAAAGGCAGAATGATGAACACA-3'.
  • the 10 targets are as follows:
  • sgRNA DQA-g1 target 5'-TTAAGCCATAGGAGGCAACA-3';
  • sgRNA DQA-g2 target 5'-GCCATAGGAGGCAACATGGT-3';
  • sgRNA DQA-g3 target 5'-CCATGAATTTGATGGCGACG-3';
  • sgRNA DQA-g4 target 5'-CCTCGTCGCCATCAAAATTCA-3';
  • sgRNA DQA-gn1 target 5'-CTGGTAGACATTTAAGCCAT-3';
  • sgRNA DQA-gn2 target 5'-GTAGACATTTAAGCCATAGG-3';
  • sgRNA DQA-gn3 target 5'-TTAAATGTCTACCAGTCTTA-3';
  • sgRNA DQA-gn4 target 5'-AGACAGTCTCCTTCTTCCCC-3';
  • sgRNA DQA-gn5 target 5'-TGGGGAAGAAGGAGACTGTC-3';
  • sgRNA DQA-gn6 target 5'-TTGACCCACAGGGTGCACTG-3'.
  • the plasmid pKG-U6gRNA was taken, digested with the restriction enzyme BbsI, and the vector backbone (a linear large fragment of about 3 kb) was recovered.
  • DQA-g1S and DQA-g1A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 11A).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (DQA-g1).
  • the plasmid pKG-U6gRNA(DQA-gl)) expresses the sgRNA DQA-gl shown in SEQ ID NO:16.
  • DQA-g2S and DQA-g2A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (Fig. 11B).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (DQA-g2).
  • the plasmid pKG-U6 gRNA (DQA-g2) expresses the sgRNA DQA-g2 shown in SEQ ID NO:17.
  • DQA-g3S and DQA-g3A were synthesized separately, then mixed and annealed to yield double-stranded DNA molecules with cohesive ends (FIG. 11C).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (DQA-g3).
  • the plasmid pKG-U6 gRNA (DQA-g3) expresses the sgRNA DQA-g3 shown in SEQ ID NO:18.
  • DQA-g4S and DQA-g4A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 11D).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (DQA-g4).
  • the plasmid pKG-U6 gRNA (DQA-g4) expresses the sgRNA DQA-g4 shown in SEQ ID NO:19.
  • DQA-gn1S and DQA-gn1A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends (FIG. 11E).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (DQA-gn1).
  • the plasmid pKG-U6 gRNA (DQA-gn1) expresses the sgRNA DQA-gn1 shown in SEQ ID NO:20.
  • DQA-gn2S and DQA-gn2A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 11F).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (DQA-gn2).
  • the plasmid pKG-U6 gRNA (DQA-gn2) expresses the sgRNA DQA-gn2 shown in SEQ ID NO:21.
  • DQA-gn3S and DQA-gn3A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends (FIG. 11G).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (DQA-gn3).
  • the plasmid pKG-U6 gRNA (DQA-gn3) expresses the sgRNA DQA-gn3 shown in SEQ ID NO:22.
  • DQA-gn4S and DQA-gn4A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 11H).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (DQA-gn4).
  • the plasmid pKG-U6 gRNA (DQA-gn4) expresses the sgRNA DQA-gn4 shown in SEQ ID NO:23.
  • DQA-gn5S and DQA-gn5A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends (Fig. 11I).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (DQA-gn5).
  • the plasmid pKG-U6 gRNA (DQA-gn5) expresses the sgRNA DQA-gn5 shown in SEQ ID NO:24.
  • DQA-gn6S and DQA-gn6A were synthesized separately, then mixed and annealed to yield double-stranded DNA molecules with cohesive ends (FIG. 11J).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (DQA-gn6).
  • the plasmid pKG-U6 gRNA (DQA-gn6) expresses the sgRNA DQA-gn6 shown in SEQ ID NO:25.
  • sgRNA-DQA-1S 5'-caccgTTAAGCCATAGGAGGCAACA-3';
  • sgRNA-DQA-1A 5'-aaacTGTTGCCTCCTATGGCTTAAc-3'.
  • sgRNA-DQA-2S 5'-caccGCCATAGGAGGCAACATGGT-3';
  • sgRNA-DQA-2A 5'-aaacACCATGTTGCCTCCTATGGC-3'.
  • sgRNA-DQA-3S 5'-caccgCCATGAATTTGATGGCGACG-3';
  • sgRNA-DQA-3A 5'-aaacCGTCGCCATCAAAATTCATGGc-3'.
  • sgRNA-DQA-4S 5'-caccgCCTCGTCGCCATCAAAATTCA-3';
  • sgRNA-DQA-4A 5'-aaacTGAATTTGATGGCGACGAGGc-3'.
  • sgRNA-DQA-n1S 5'-caccgCTGGTAGACATTTAAGCCAT-3';
  • sgRNA-DQA-n1A 5'-aaacATGGCTTAAATGTCTACCAGc-3'.
  • sgRNA-DQA-n2S 5'-caccGTAGACATTTAAGCCATAGG-3';
  • sgRNA-DQA-n2A 5'-aaacCCTATGGCTTAAATGTCTAC-3'.
  • sgRNA-DQA-n3S 5'-caccgTTAAATGTCTACCAGTCTTA-3';
  • sgRNA-DQA-n3A 5'-aaacTAAGACTGGTAGACATTTAAc-3'.
  • sgRNA-DQA-n4S 5'-caccgAGACAGTCTCCTTCTTCCCC-3';
  • sgRNA-DQA-n4A 5'-aaacGGGGAAGAAGGAGACTGTCTc-3'.
  • sgRNA-DQA-n5S 5'-caccgTGGGGAAGAAGGAGACTGTC-3';
  • sgRNA-DQA-n5A 5'-aaacGACAGTCTCCTTCTTCCCCAc-3'.
  • sgRNA-DQA-n6S 5'-caccgTTGACCCACAGGGTGCACTG-3';
  • sgRNA-DQA-n6A 5'-aaacCAGTGCACCCTGTGGGTCAAc-3'.
  • Group 1 Plasmid pKG-U6 gRNA (DQA-gl) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DQA-g1): 1.238 ⁇ g plasmid pKG-GE3.
  • the second group the plasmid pKG-U6 gRNA (DQA-g2) and the plasmid pKG-GE3 were co-transfected into primary porcine fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6 gRNA (DQA-g2): 1.238 ⁇ g plasmid pKG-GE3.
  • the third group the plasmid pKG-U6gRNA (DQA-g3) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts.
  • Ratio about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DQA-g3): 1.238 ⁇ g plasmid pKG-GE3.
  • the fourth group co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (DQA-g4) and plasmid pKG-GE3.
  • Ratio about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DQA-g4): 1.238 ⁇ g plasmid pKG-GE3.
  • the fifth group co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (DQA-gn1) and plasmid pKG-GE3.
  • Ratio about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DQA-gn1): 1.238 ⁇ g plasmid pKG-GE3.
  • the sixth group co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (DQA-gn2) and plasmid pKG-GE3.
  • Ratio about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DQA-gn2): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 7 Co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (DQA-gn3) and plasmid pKG-GE3. Ratio: about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DQA-gn3): 1.238 ⁇ g plasmid pKG-GE3.
  • the eighth group Co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (DQA-gn4) and plasmid pKG-GE3. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DQA-gn4): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 9 Co-transfection of plasmid pKG-U6 gRNA (DQA-gn5) and plasmid pKG-GE3 into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DQA-gn5): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 10 Co-transfection of plasmid pKG-U6 gRNA (DQA-gn6) and plasmid pKG-GE3 into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6 gRNA (DQA-gn6): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 11 porcine primary fibroblasts without any transfection.
  • Co-transfection was performed by electroporation using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use the complete culture medium to culture for 16-18 hours, and then replace with a new complete culture medium for culture. The total incubation time was 48 hours.
  • step 2 the cells were digested with trypsin and collected, the genomic DNA was extracted, PCR amplification was carried out using the primer pair composed of DQA-F643 and DQA-R1022, and then electrophoresis and sequencing were performed. The results are shown in Figure 12.
  • the editing efficiency of different targets was obtained by analyzing the sequencing peak map using the Synthego ICE tool.
  • the editing efficiencies of the first to eleventh groups of different targets were 35%, 29%, 11%, 0%, 12%, 44%, 11%, 0%, 12%, 40% and 0%.
  • the results showed that the sixth group had the highest editing efficiency, and sgRNA DQA-gn2 was the optimal target.
  • Pig DRA gene information encoding MHC class II DR-alpha precursor; located on chromosome 7; GeneID is 100135040, Sus scrofa.
  • the protein encoded by the porcine DRA gene is shown in SEQ ID NO: 26 (exemplary sequence in NCBI).
  • SEQ ID NO: 27 the sequencing result of Congjiangxiang pig in the present invention.
  • the genomic DNAs of 8 pigs were used as templates, and the primer pair consisting of primers DRA-GT-F326/DRA-GT-R1192 was used for PCR amplification, and then electrophoresis was performed, as shown in FIG. 13 .
  • the PCR amplification products were recovered and sequenced, and the sequencing results were compared and analyzed with the DRA gene sequences in the public database. Based on the alignment results, primers were designed to detect mutations (primers themselves avoid possible mutation sites).
  • the primers designed to detect mutations are: DRA-F573/DRA-R968.
  • DRA-GT-F326 5'-TTTCACGGACAGTCACATGGAGT-3';
  • DRA-GT-R1192 5'-ATACCTAGCTCTGAAATCCGCCC-3'.
  • DRA-F573 5'-TCATCGCCTTCTCTATTTTCCAC-3';
  • DRA-R968 5'-CCCCTGGAAGGAAAAGTAAGTCA-3'.
  • the 4 targets are as follows:
  • sgRNA DRA-g1 target 5'-TCCACGTGGATATGGAAAAG-3';
  • sgRNA DRA-g2 target 5'-CCCTCTTTTCCATATCCACG-3';
  • sgRNA DRA-g3 target 5'-AGCTGTGGACAAAGCCAACC-3';
  • sgRNA DRA-g4 target 5'-TGCACCCTGAGCCTCAAAGC-3'.
  • the plasmid pKG-U6gRNA was taken, digested with the restriction enzyme BbsI, and the vector backbone (a linear large fragment of about 3 kb) was recovered.
  • DRA-g1S and DRA-g1A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 14A).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (DRA-g1).
  • the plasmid pKG-U6 gRNA (DRA-gl) expresses the sgRNA DRA-gl shown in SEQ ID NO:28.
  • DRA-g2S and DRA-g2A were synthesized separately, then mixed and annealed to yield double-stranded DNA molecules with cohesive ends (FIG. 14B).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (DRA-g2).
  • the plasmid pKG-U6 gRNA (DRA-g2) expresses the sgRNA DRA-g2 shown in SEQ ID NO:29.
  • DRA-g3S and DRA-g3A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 14C).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (DRA-g3).
  • the plasmid pKG-U6 gRNA (DRA-g3) expresses the sgRNA DRA-g3 shown in SEQ ID NO:30.
  • DRA-g4S and DRA-g4A were synthesized separately, then mixed and annealed to yield double-stranded DNA molecules with cohesive ends (FIG. 14D).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (DRA-g4).
  • Plasmid pKG-U6 gRNA (DRA-g4) expresses the sgRNA DRA-g4 shown in SEQ ID NO:31.
  • sgRNA-DRA-1S 5'-caccgTCCACGTGGATATGGAAAAG-3';
  • sgRNA-DRA-1A 5'-aaacCTTTTCCATATCCACGTGGAc-3'.
  • sgRNA-DRA-2S 5'-caccgCCCTCTTTTCCATATCCACG-3';
  • sgRNA-DRA-2A 5'-aaacCGTGGATATGGAAAAGAGGGc-3'.
  • sgRNA-DRA-3S 5'-caccgAGCTGTGGACAAAGCCAACC-3';
  • sgRNA-DRA-3A 5'-aaacGGTTGGCTTTGTCCACAGCTc-3'.
  • sgRNA-DRA-4S 5'-caccgTGCACCCTGAGCCTCAAAGC-3';
  • sgRNA-DRA-4A 5'-aaacGCTTTGAGGCTCAGGGTGCAc-3'.
  • sgRNA-DRA-1S, sgRNA-DRA-1A, sgRNA-DRA-2S, sgRNA-DRA-2A, sgRNA-DRA-3S, sgRNA-DRA-3A, sgRNA-DRA-4S, sgRNA-DRA-4A are single Stranded DNA molecule.
  • Group 1 Plasmid pKG-U6 gRNA (DRA-g1) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DRA-g1): 1.238 ⁇ g plasmid pKG-GE3.
  • the second group the plasmid pKG-U6 gRNA (DRA-g2) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts.
  • Ratio about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DRA-g2): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 3 Co-transfection of plasmid pKG-U6 gRNA (DRA-g3) and plasmid pKG-GE3 into primary porcine fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DRA-g3): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 4 Co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (DRA-g4) and plasmid pKG-GE3. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (DRA-g4): 1.238 ⁇ g plasmid pKG-GE3.
  • the fifth group porcine primary fibroblasts without any transfection.
  • Co-transfection was performed by electroporation, using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use the complete culture medium to culture for 16-18 hours, and then replace with a new complete culture medium for culture. The total incubation time was 48 hours.
  • step 2 the cells were digested with trypsin and collected, the genomic DNA was extracted, PCR amplification was carried out using the primer pair composed of DRA-F573 and DRA-R968, and then electrophoresis and sequencing were performed. The results are shown in Figure 15.
  • the editing efficiency of different targets was obtained by analyzing the sequencing peak map using the Synthego ICE tool.
  • the editing efficiencies of the first to fifth groups of different targets were 52%, 45%, 24%, 20% and 0%, respectively.
  • the results showed that the editing efficiency of the first group was the highest, and sgRNA DRA-g1 was the optimal target.
  • Pig IL2RG gene information encoding interleukin 2receptor subunit gamma; located on the X chromosome;
  • GeneID is 397156, Sus scrofa.
  • the protein encoded by the porcine IL2RG gene is shown in SEQ ID NO:32.
  • the porcine IL2RG gene has 9 exons, of which the 4th exon and its upstream and downstream sequences of 500 bp are shown in SEQ ID NO: 33.
  • the genomic DNAs of 8 pigs were used as templates, and the primer pair consisting of primers IL2RG-GT-F4543/IL2RG-GT-R5180 was used for PCR amplification, and then electrophoresis was performed, as shown in Figure 16.
  • the PCR amplification products were recovered and sequenced, and the sequencing results were compared and analyzed with the IL2RG gene sequence in the public database. Based on the alignment results, primers for detecting mutations (primers themselves avoid possible mutation sites) are designed.
  • the primers designed to detect mutations are: IL2RG-nF33/IL2RG-nR460.
  • IL2RG-GT-F4543 5'-ATATAGCACAGGGGAGGGAGGAA-3';
  • IL2RG-GT-R5180 5'-AGGGTGCGAAGGGTCAGATTC-3';
  • IL2RG-nF33 5'-CCCAGGCTTCCCACTATATTCTC-3';
  • IL2RG-nR460 5'-CCATTGGATCCCTCACTTCTTCT-3'.
  • the 9 targets are as follows:
  • sgRNA IL2RG-g1 target 5'-CCTGTAGTTTTAGCGTCTGT-3';
  • sgRNA IL2RG-g2 target 5'-CAACAAATTTTTGGTAGAGG-3';
  • sgRNA IL2RG-g3 target 5'-GATGATAAAGTCCAGGAGTG-3';
  • sgRNA IL2RG-g4 target 5'-CTGGACTTTATCATCATTAG-3';
  • sgRNA IL2RG-g5 target 5'-TTGTCCAGCTCCAGGACCCA-3';
  • sgRNA IL2RG-g6 target 5'-GGCCACTATCTATTCTCTGA-3';
  • sgRNA IL2RG-g7 target 5'-TCCCTTCAGAGAATAGATAG-3';
  • sgRNA IL2RG-g8 target 5'-AACATTTGTTGTCCAGCTCC-3';
  • sgRNA IL2RG-g9 target 5'-TGTCCAGCTCCAGGACCCAC-3'.
  • the plasmid pKG-U6gRNA was taken, digested with the restriction enzyme BbsI, and the vector backbone (a linear large fragment of about 3 kb) was recovered.
  • IL2RG-g1S and IL2RG-g1A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 17A).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (IL2RG-g1).
  • the plasmid pKG-U6 gRNA (IL2RG-gl) expresses the sgRNA IL2RG-gl shown in SEQ ID NO:34.
  • IL2RG-g2S and IL2RG-g2A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 17B).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (IL2RG-g2).
  • the plasmid pKG-U6 gRNA (IL2RG-g2) expresses the sgRNA IL2RG-g2 shown in SEQ ID NO:35.
  • IL2RG-g3S and IL2RG-g3A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 17C).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (IL2RG-g3).
  • the plasmid pKG-U6 gRNA (IL2RG-g3) expresses the sgRNA IL2RG-g3 shown in SEQ ID NO:36.
  • IL2RG-g4S and IL2RG-g4A were synthesized separately, then mixed and annealed to yield double-stranded DNA molecules with cohesive ends (FIG. 17D).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (IL2RG-g4).
  • the plasmid pKG-U6 gRNA (IL2RG-g4) expresses the sgRNA IL2RG-g4 shown in SEQ ID NO:37.
  • IL2RG-g5S and IL2RG-g5A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 17E).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (IL2RG-g5).
  • the plasmid pKG-U6 gRNA (IL2RG-g5) expresses the sgRNA IL2RG-g5 shown in SEQ ID NO:38.
  • IL2RG-g6S and IL2RG-g6A were synthesized separately, then mixed and annealed, resulting in double-stranded DNA molecules with cohesive ends (FIG. 17F).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6 gRNA (IL2RG-g6).
  • the plasmid pKG-U6 gRNA (IL2RG-g6) expresses the sgRNA IL2RG-g6 shown in SEQ ID NO:39.
  • IL2RG-g7S and IL2RG-g7A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends (FIG. 17G).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (IL2RG-g7).
  • the plasmid pKG-U6 gRNA (IL2RG-g7) expresses the sgRNA IL2RG-g7 shown in SEQ ID NO:40.
  • IL2RG-g8S and IL2RG-g8A were synthesized separately, then mixed and annealed to yield double-stranded DNA molecules with cohesive ends (FIG. 17H).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (IL2RG-g8).
  • the plasmid pKG-U6 gRNA (IL2RG-g8) expresses the sgRNA IL2RG-g8 shown in SEQ ID NO:41.
  • IL2RG-g9S and IL2RG-g9A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends (FIG. 17I).
  • the double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (IL2RG-g9).
  • the plasmid pKG-U6 gRNA (IL2RG-g9) expresses the sgRNA IL2RG-g9 shown in SEQ ID NO:42.
  • sgRNA-IL2RG-1S 5'-caccgCCTGTAGTTTTAGCGTCTGT-3';
  • sgRNA-IL2RG-1A 5'-aaacACAGACGCTAAAACTACAGGc-3'.
  • sgRNA-IL2RG-2S 5'-caccgCAACAAATTTTTGGTAGAGG-3';
  • sgRNA-IL2RG-2A 5'-aaacCCCTTACCAAACATTTGTTGc-3'.
  • sgRNA-IL2RG-3S 5'-caccGATGATAAAGTCCAGGAGTG-3';
  • sgRNA-IL2RG-3A 5'-aaacCACTCCTGGACTTTATCATC-3'.
  • sgRNA-IL2RG-4S 5'-caccgCTGGACTTTATCATCATTAG-3';
  • sgRNA-IL2RG-4A 5'-aaacCTAATGATGATAAAGTCCAGc-3'.
  • sgRNA-IL2RG-5S 5'-caccgTTGTCCAGCTCCAGGACCCA-3';
  • sgRNA-IL2RG-5A 5'-aaacTGGGTCCTGGAGCTGGACAAc-3'.
  • sgRNA-IL2RG-6S 5'-caccgGGCCACTATCTATTCTCTGA-3';
  • sgRNA-IL2RG-6A 5'-aaacTCAGAGAATAGATAGTGGCCc-3'.
  • sgRNA-IL2RG-7S 5'-caccgTCCCTTCAGAGAATAGATAG-3';
  • sgRNA-IL2RG-7A 5'-aaacCTATCTATTCTCTGAAGGGAc-3'.
  • sgRNA-IL2RG-8S 5'-caccgAACATTTGTTGTCCAGCTCC-3';
  • sgRNA-IL2RG-8A 5'-aaacGGAGCTGGACAACAAATGTTc-3'.
  • sgRNA-IL2RG-9S 5'-caccgTGTCCAGCTCCAGGACCCAC-3';
  • sgRNA-IL2RG-9A 5'-aaacGTGGGTCCTGGAGCTGGAACAc-3'.
  • Group 1 Plasmid pKG-U6 gRNA (IL2RG-g1) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (IL2RG-g1): 1.238 ⁇ g plasmid pKG-GE3.
  • the second group the plasmid pKG-U6gRNA (IL2RG-g2) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (IL2RG-g2): 1.238 ⁇ g plasmid pKG-GE3.
  • the third group the plasmid pKG-U6gRNA (IL2RG-g3) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts.
  • Ratio about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (IL2RG-g3): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 4 Co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (IL2RG-g4) and plasmid pKG-GE3. Ratio: about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (IL2RG-g4): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 5 Co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (IL2RG-g5) and plasmid pKG-GE3. Ratio: about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (IL2RG-g5): 1.238 ⁇ g plasmid pKG-GE3.
  • the sixth group co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (IL2RG-g6) and plasmid pKG-GE3.
  • Ratio about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (IL2RG-g6): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 7 Plasmid pKG-U6 gRNA (IL2RG-g7) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (IL2RG-g7): 1.238 ⁇ g plasmid pKG-GE3.
  • the eighth group co-transfected porcine primary fibroblasts with plasmid pKG-U6 gRNA (IL2RG-g8) and plasmid pKG-GE3.
  • Ratio about 200,000 primary porcine fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (IL2RG-g8): 1.238 ⁇ g plasmid pKG-GE3.
  • the ninth group the plasmid pKG-U6 gRNA (IL2RG-g9) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts.
  • Ratio about 200,000 porcine primary fibroblasts: 0.762 ⁇ g plasmid pKG-U6gRNA (IL2RG-g9): 1.238 ⁇ g plasmid pKG-GE3.
  • Group 10 porcine primary fibroblasts without any transfection.
  • Co-transfection was performed by electroporation, using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use the complete culture medium to culture for 16-18 hours, and then replace with a new complete culture medium for culture. The total incubation time was 48 hours.
  • step 3 trypsinize and collect cells, extract genomic DNA, perform PCR amplification with primer pairs composed of IL2RG-nF33 and IL2RG-nR460, and then perform electrophoresis and sequencing. The results are shown in Figure 18.
  • the editing efficiency of different targets was obtained by analyzing the sequencing peak map using the Synthego ICE tool.
  • the editing efficiencies of the first to tenth groups of different targets were 1%, 0%, 3%, 5%, 0%, 46%, 65%, 18%, 34% and 0%.
  • the results showed that the seventh group had the highest editing efficiency, and sgRNA IL2RG-g7 was the optimal target.
  • Plasmid pKG-U6gRNA (ADA-g7), plasmid pKG-U6gRNA (DQA-gn2), plasmid pKG-U6gRNA (DRA-g1), plasmid pKG-U6gRNA (IL2RG-g7) and plasmid pKG-GE3 were co-transfected into primary pigs Fibroblasts.
  • Ratio about 200,000 porcine primary fibroblasts: 0.34 ⁇ g plasmid pKG-U6gRNA (ADA-g7): 0.34 ⁇ g plasmid pKG-U6gRNA (DQA-gn2): 0.34 ⁇ g plasmid pKG-U6gRNA (DRA-g1): 0.34 ⁇ g of plasmid pKG-U6 gRNA (IL2RG-g7): 1.64 ⁇ g of plasmid pKG-GE3.
  • Co-transfection was performed by electroporation, using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use the complete culture medium to culture for 16-18 hours, and then replace with a new complete culture medium for culture. The total incubation time was 48 hours.
  • step 3 trypsinize and collect cells, then wash with complete culture medium, then resuspend with complete culture medium, and then pick each monoclonal and transfer it to a 96-well plate (1 cell per well). , each well was filled with 200 ⁇ l of complete medium), and cultured for 2 weeks (replace with new complete medium every 2-3 days).
  • step 3 trypsinize and collect the cells (about 2/3 of the cells obtained in each well are seeded into a 6-well plate with complete culture medium, and the remaining 1/3 is collected in a 1.5 mL centrifuge tube. ).
  • step 5 Take the 6-well plate from step 4, culture until the cells grow to 50% fullness, digest and collect the cells with trypsin, and freeze the cells with cell freezing solution (90% complete medium + 10% DMSO, by volume). live.
  • step 6 Take the centrifuge tube of step 4, take the cells, extract the genomic DNA, and carry out PCR amplification (respectively use the primer pair composed of ADA-nnF229 and ADA-nnR456, the primer pair composed of DQA-F643 and DQA-R1022, DRA-F573 A primer pair consisting of DRA-R968, a primer pair consisting of IL2RG-nF33 and IL2RG-nR460), followed by electrophoresis. Pig primary fibroblasts were used as wild-type controls.
  • Figure 19 shows the electropherogram of the primer pair composed of ADA-nnF229 and ADA-nnR456.
  • Figure 20 shows the electropherogram of the primer pair composed of DQA-F643 and DQA-R1022.
  • Figure 21 shows the electropherogram of the primer pair consisting of DRA-F573 and DRA-R968.
  • Figure 22 shows the electropherogram of the primer pair consisting of IL2RG-nF33 and IL2RG-nR460.
  • step 6 the PCR amplification product is recovered and sequenced.
  • porcine primary fibroblasts There is only one sequencing result of porcine primary fibroblasts, and its genotype is homozygous wild type. If there are two kinds of sequencing results of a monoclonal cell, one is consistent with the sequencing results of porcine primary fibroblasts, and the other is mutated compared with the sequencing results of porcine primary fibroblasts (mutations include one or more nucleotide deletion, insertion or substitution), the genotype of the monoclonal cell is heterozygous; if the sequencing result of a monoclonal cell is two, compared with the sequencing result of primary porcine fibroblasts.
  • Mutation includes deletion, insertion or substitution of one or more nucleotides
  • the genotype of the monoclonal cell is a biallelic different mutant; if the sequencing result of a monoclonal cell is one, and the Compared with the sequencing results of porcine primary fibroblasts, there is a mutation (mutation includes deletion, insertion or substitution of one or more nucleotides), and the genotype of the monoclonal cell is the same biallelic mutant; if a certain The sequencing result of the monoclonal cell is one, which is consistent with the sequencing result of the primary fibroblast of the pig, and the genotype of the monoclonal cell is homozygous wild type.
  • the editing results of the ADA gene are shown in Table 1.
  • the genotypes of the monoclonal cells numbered 31 and 43 were biallelic identical mutants.
  • the genotypes of the monoclonal cells numbered 45 and 52 were biallelic different mutants.
  • the genotype of the monoclonal cell numbered 39 was heterozygous.
  • the monoclonal cells numbered 4, 6, 12, 14, 20, 28, 29, 34, 48, and 50 all showed complex sets of peaks, so an effective sequence could not be obtained, and the genotype and specific form could not be determined, but It can be judged that gene editing has occurred.
  • the resulting ratio of gene-edited monoclonal cells was 15/73.
  • An exemplary ADA sequencing peak profile is shown in Figure 23.
  • the editing results of the DQA gene are shown in Table 2.
  • the genotypes of the monoclonal cells numbered 29, 31, 32, 45, 51, and 69 were biallelic identical mutants.
  • the genotypes of the monoclonal cells numbered 39, 52, and 61 were biallelic different mutants.
  • the genotypes of monoclonal cells numbered 44, 70, and 72 were heterozygous.
  • the monoclonal cells numbered 4, 6, 12, 14, 20, 28, 34, 46, and 67 all showed complex sets of peaks, so the effective sequence could not be obtained, and the genotype and specific form could not be determined, but it could be judged Gene editing has occurred.
  • the resulting ratio of DQA gene-edited monoclonal cells was 21/71.
  • An exemplary DQA sequencing peak graph is shown in Figure 24.
  • the editing results of the DRA gene are shown in Table 3.
  • the genotypes of monoclonal cells numbered 31 and 50 were biallelic identical mutants.
  • the genotype of monoclonal cell number 69 was heterozygous.
  • the monoclonal cells numbered 6, 12, 29, 45, and 52 all showed complex sets of peaks, so effective sequences could not be obtained, and the genotype and specific form could not be determined, but gene editing could be judged.
  • the ratio of DRA gene-edited monoclonal cells was 8/71.
  • An exemplary DRA sequencing peak profile is shown in Figure 25.
  • the editing results of the IL2RG gene are shown in Table 4.
  • the genotypes of the monoclonal cells numbered 12 and 61 were biallelic identical mutants.
  • the genotypes of monoclonal cells numbered 43, 45, and 50 were biallelic different mutants.
  • the genotypes of the monoclonal cells numbered 46, 48, 51, 69 and 70 were heterozygous.
  • the monoclonal cells numbered 6, 20, 28, 29, 31, 32, and 52 all showed complex sets of peaks, so the effective sequence could not be obtained, and the genotype and specific form could not be determined, but it could be judged that gene editing occurred .
  • the resulting ratio of IL2RG gene-edited monoclonal cells was 17/72.
  • An exemplary sequencing peak profile of IL2RG is shown in Figure 26.
  • step 7 select monoclonal cells in which IL2RG, ADA, DQA and DRA genes are simultaneously knocked out.
  • the monoclonal cells numbered 6, 12, 29, 31, 45, and 52 were the monoclonal cells with simultaneous knockout of ADA, DQA, DRA and IL2RG genes.
  • the present invention can be used to obtain a severe immunodeficiency pig model by means of gene editing, and can be used for drug screening, drug efficacy detection, disease pathology, gene therapy, cell therapy and other researches, and can provide effective experimental data for further clinical applications, and also provides In the future, it will provide a powerful experimental means to cure severe human immunodeficiency.
  • the invention lays a solid foundation for the preparation of a severe immunodeficiency pig model, and has great application value for the research and development of a severe immunodeficiency drug.

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Abstract

提供了一种CRISPR系统及其在制备多基因联合敲除的重症免疫缺陷克隆猪核供体细胞中的应用。提供了一种制备ADA基因、DQA基因、DRA基因和IL2RG基因这四个基因联合敲除的重症免疫缺陷猪源重组细胞的方法、CRISPR系统、sgRNA组合、质粒组合和试剂盒，所述sgRNA组合由SEQ ID NO：11所示sgRNA ADA-g7、SEQ ID NO：21所示sgRNA DQA-gn2、SEQ ID NO：28所示sgRNA DRA-g1和SEQ ID NO：40所示sgRNA IL2RG-g7组成，该sgRNA组合可用于制备重组细胞、免疫缺陷动物模型和免疫缺陷动物细胞模型。

Description

CRISPR系统及其在制备多基因联合敲除的重症免疫缺陷克隆猪核供体细胞中的应用 技术领域
本发明涉及CRISPR系统及其在制备多基因联合敲除的重症免疫缺陷克隆猪核供体细胞中的应用，具体涉及一种制备ADA基因、DQA基因、DRA基因和IL2RG基因这四个基因联合敲除的重症免疫缺陷猪源重组细胞的方法、CRISPR系统、sgRNA组合、质粒组合和试剂盒。
背景技术
重症联合免疫缺陷(severe combined immunodeficiency,SCID)是原发性免疫缺陷病中最严重的表型，是指由于遗传、发育或感染等因素导致的T细胞、B细胞和NK细胞同时出现发育、分化、增值、代谢或功能障碍。1950年，Glanzmann和Riniker首次报道了人类婴儿中的SCID病。在全球，SCID的新生儿发病率约为1/50000，该病发病年龄早，临床表现重，且死亡率高。多数SCID是由于免疫相关基因的异常造成的，而SCID的主要遗传方式包括X连锁隐性遗传和常染色体隐性遗传两种。该病发病具有一定的区域性和血缘性，且由于存在X连锁隐性遗传的特性，该病多见于男性患者。
目前，针对于SCID的治疗手段主要包括骨髓或造血干细胞移植和基因治疗。骨髓或干细胞移植是治疗SCID的最佳方案，但是寻找与患者合适配型的供体相当困难。
由常染色体隐性遗传导致的SCID，又分为核苷酸代谢相关酶缺陷和主要组织相容性复合体分子缺陷(major histocompatibility complex,MHC)引起的SCID。核苷酸代谢相关酶中的嘌呤核苷酸化酶相关基因ADA的缺陷会导致细胞内核苷酸代谢产物dATP或dGTP的大量富集，这些产物对淋巴细胞具有选择毒性作用，从而造成淋巴细胞的功能障碍、受损或死亡，进而引发SCID。主要组织相容性复合体分子缺陷导致的SCID是因6号染色体短臂的MHC异常所致，其又分为MHC I类和MHC II类缺陷。I类为TAP基因缺陷造成MHC I类分子结构不稳定所致；II类为DQA、DRA等基因缺陷造成MHC II类分子不表达或低表达所致。MHC分子的功能故障会导致T细胞无法识别抗原信号，从而引起其相关的免疫应答缺失，进而引发SCID。
X连锁隐性遗传导致的SCID是最常见的一种类型，其致病突变为编码IL-2Rγ链的IL2RG基因发生突变，从而导致IL-2Rγ链功能发生障碍。而IL-2Rγ链也被称为共有γ链(common gamma chain)，是IL-2、IL-4和IL-7等多种参与调控免疫细胞分化、发育、成熟过程的细胞因子受体与其相应配体结合后，向免疫细胞内部转导信号时所共同使用的信号转导分子。因此，共有γ链的功能障碍会导致免疫细胞的功能或发育异常，从而引发SCID。
作为大动物，猪是人类长期以来主要的肉食来源动物，易于大规模繁殖饲养，而且在伦理道德及动物保护等方面的要求较低，同时猪体型大小和器官功能与人类近似，是理想的人类疾病模型动物。另外，在进行研究生物活性大分子或细胞治疗的效果时，用异源动物进行试验将会产生免疫排斥，从而无法进行有效的动物试验。 而用重症联合免疫缺陷模式动物则可避免异种间的免疫排斥问题。因此，研发出人类SCID猪模型用于进行药物(特别是生物活性分子)筛选、药效检测、疾病病理、基因及细胞治疗等研究，能够为进一步的临床应用提供有效的实验数据，也为成功治疗人类SCID疾病提供有力的实验手段。
发明公开
本发明涉及CRISPR系统及其在制备多基因联合敲除的重症免疫缺陷克隆猪核供体细胞中的应用，具体涉及一种制备ADA基因、DQA基因、DRA基因和IL2RG基因这四个基因联合敲除的重症免疫缺陷猪源重组细胞的方法、CRISPR系统、sgRNA组合、质粒组合和试剂盒。
本发明提供了一种制备重组细胞的方法，包括如下步骤：将质粒pKG-T6gRNA(ADA-g7)、质粒pKG-T6gRNA(DQA-gn2)、质粒pKG-T6gRNA(DRA-g1)、质粒pKG-T6gRNA(IL2RG-g7)和质粒pKG-GE3共转染猪细胞，得到ADA基因、DQA基因、DRA基因和IL2RG基因均发生突变的重组细胞。所述猪细胞可为猪成纤维细胞。所述猪细胞具体可为猪原代成纤维细胞。所述猪具体可为从江香猪。
所述方法制备得到的重组细胞也属于本发明的保护范围。
具体来说，所述重组细胞可为如下任一：表1至表4中编号为6、12、29、31、45、52的单克隆细胞株。
本发明还保护所述重组细胞在制备免疫缺陷动物模型中的应用。本发明还保护所述重组细胞在制备免疫缺陷动物细胞模型中的应用。制备免疫缺陷动物模型时，将所述重组细胞作为核移植供体细胞采用体细胞克隆技术得到克隆猪，即为免疫缺陷动物模型。可以用免疫缺陷动物模型制备免疫缺陷动物细胞模型，即分离免疫缺陷动物模型的相应细胞，作为免疫缺陷动物细胞模型。所述动物模型为猪模型。所述动物细胞模型为猪细胞模型。所述动物为猪，具体可为从江香猪。
本发明还提供了sgRNA组合，由sgRNA ADA-g7、sgRNA DQA-gn2、sgRNA DRA-g1和sgRNA IL2RG-g7组成。
本发明还提供了质粒组合，由质粒pKG-T6gRNA(ADA-g7)、质粒pKG-T6gRNA(DQA-gn2)、质粒pKG-T6gRNA(DRA-g1)和质粒pKG-T6gRNA(IL2RG-g7)组成。
本发明还提供了一种试剂盒，包括所述sgRNA组合。
本发明还提供了一种试剂盒，包括所述质粒组合。所述试剂盒还包括质粒pKG-GE3。
本发明还保护所述sgRNA组合在制备试剂盒中的应用。
本发明还保护所述质粒组合在制备试剂盒中的应用。
本发明还保护所述质粒组合和质粒pKG-GE3在制备试剂盒中的应用。
以上任一所述试剂盒的用途为如下(a)或(b)或(c)：(a)制备重组细胞；(b)制备免疫缺陷动物模型；(c)制备免疫缺陷动物细胞模型。制备免疫缺陷动物模型时，先制备所述重组细胞，然后将所述重组细胞作为核移植供体细胞采用体细胞克隆技术得到克隆动物，即为免疫缺陷动物模型。可以用免疫缺陷动物模 型制备免疫缺陷动物细胞模型，即分离免疫缺陷动物模型的相应细胞，作为免疫缺陷动物细胞模型。所述动物具体可为猪。所述动物模型为猪模型。所述动物细胞模型为猪细胞模型。所述重组细胞为猪重组细胞。所述重组细胞的转化受体细胞为猪细胞。所述猪细胞可为猪成纤维细胞。所述猪细胞具体可为猪原代成纤维细胞。所述猪具体可为从江香猪。
本发明还保护以上任一所述sgRNA组合或以上任一所述质粒组合或以上任一所述试剂盒在制备重组细胞中的应用。所述重组细胞为猪重组细胞。所述重组细胞的转化受体细胞为猪细胞。所述猪细胞可为猪成纤维细胞。所述猪细胞具体可为猪原代成纤维细胞。所述猪具体可为从江香猪。
本发明还保护以上任一所述sgRNA组合或以上任一所述质粒组合或以上任一所述试剂盒在制备免疫缺陷动物模型中的应用。本发明还保护以上任一所述sgRNA组合或以上任一所述质粒组合或以上任一所述试剂盒在制备免疫缺陷动物细胞模型中的应用。应用时，先制备所述重组细胞，然后将所述重组细胞作为核移植供体细胞采用体细胞克隆技术得到克隆动物，即为免疫缺陷动物模型。可以用免疫缺陷动物模型制备免疫缺陷动物细胞模型，即分离免疫缺陷动物模型的相应细胞，作为免疫缺陷动物细胞模型。所述动物模型为猪模型。所述动物细胞模型为猪细胞模型。所述动物具体可为猪。所述重组细胞为猪重组细胞。所述重组细胞的转化受体细胞为猪细胞。所述猪细胞可为猪成纤维细胞。所述猪细胞具体可为猪原代成纤维细胞。所述猪具体可为从江香猪。
以上任一所述重组细胞为ADA基因、DQA基因、DRA基因和IL2RG基因均缺陷的细胞。
以上任一所述重组细胞为ADA基因、DQA基因、DRA基因和IL2RG基因均发生突变的重组细胞。所述突变可为杂合突变(对应基因型为杂合突变型)或纯合突变(对应的基因型为双等位基因相同突变型或双等位基因不同突变型)。
sgRNA ADA-g7靶点：5’-GGAGGGCGTGGTGTACGTGG-3’。
sgRNA DQA-gn2靶点：5’-GTAGACATTTAAGCCATAGG-3’。
gRNA DRA-g1靶点：5’-TCCACGTGGATATGGAAAAG-3’。
sgRNA IL2RG-g7靶点：5’-TCCCTTCAGAGAATAGATAG-3’。
所述质粒pKG-T6gRNA(ADA-g7)转录得到sgRNA ADA-g7。
所述质粒pKG-T6gRNA(DQA-gn2)转录得到sgRNA DQA-gn2。
所述质粒pKG-T6gRNA(DRA-g1)转录得到sgRNA DRA-g1。
所述质粒pKG-T6gRNA(IL2RG-g7)转录得到sgRNA IL2RG-g7。
所述sgRNA ADA-g7的靶序列结合区如SEQ ID NO：11中第1-20位核苷酸所示。
所述sgRNA DQA-gn2的靶序列结合区如SEQ ID NO：21中第1-20位核苷酸所示。
所述sgRNA DRA-g1的靶序列结合区如SEQ ID NO：28中第1-20位核苷酸所示。
所述sgRNA IL2RG-g7的靶序列结合区如SEQ ID NO：40中第1-20位核苷酸所示。
所述sgRNA ADA-g7如SEQ ID NO：11所示。
所述sgRNA DQA-gn2如SEQ ID NO：21所示。
所述sgRNA DRA-g1如SEQ ID NO：28所示。
所述sgRNA IL2RG-g7如SEQ ID NO：40所示。
具体来说，所述质粒pKG-T6gRNA(ADA-g7)借助限制性内切酶BbsI将sgRNA ADA-g7的靶序列结合区的编码序列插入pKG-U6gRNA载体得到的。
具体来说。所述质粒pKG-T6gRNA(DQA-gn2)是借助限制性内切酶BbsI将sgRNA DQA-gn2的靶序列结合区的编码序列插入pKG-U6gRNA载体得到的。
具体来说，所述质粒pKG-T6gRNA(DRA-g1)借助限制性内切酶BbsI将sgRNA DRA-g1的靶序列结合区的编码序列插入pKG-U6gRNA载体得到的。
具体来说。所述质粒pKG-T6gRNA(IL2RG-g7)是借助限制性内切酶BbsI将sgRNA IL2RG-g7的靶序列结合区的编码序列插入pKG-U6gRNA载体得到的。
质粒pKG-GE3中，具有特异融合基因；所述特异融合基因编码特异融合蛋白；
所述特异融合蛋白自N端至C端依次包括如下元件：两个核定位信号(NLS)、Cas9蛋白、两个核定位信号、自剪切多肽P2A、荧光报告蛋白、自裂解多肽T2A、抗性筛选标记蛋白；
质粒pKG-GE3中，由EF1a启动子启动所述特异融合基因的表达；
质粒pKG-GE3中，所述特异融合基因下游具有WPRE序列元件、3’LTR序列元件和bGH poly(A)signal序列元件。
质粒pKG-GE3中，依次具有如下元件：CMV增强子、EF1a启动子、所述特异融合基因、WPRE序列元件、3’LTR序列元件、bGH poly(A)signal序列元件。
所述特异融合蛋白中，Cas9蛋白上游的两个核定位信号为SV40核定位信号，Cas9蛋白下游的两个核定位信号为nucleoplasmin核定位信号。
所述特异融合蛋白中，荧光报告蛋白具体可为EGFP蛋白。
所述特异融合蛋白中，抗性筛选标记蛋白具体可为Puromycin蛋白。
自剪切多肽P2A的氨基酸序列为“ATNFSLLKQAGDVEENPGP”(发生自剪切的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间)。
自裂解多肽T2A的氨基酸序列为“EGRGSLLTCGDVEENPGP”(发生自裂解的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间)。
特异融合基因具体如SEQ ID NO：2中第911-6706位核苷酸所示。
CMV增强子如SEQ ID NO：2中第395-680位核苷酸所示。
EF1a启动子如SEQ ID NO：2中第682-890位核苷酸所示。
WPRE序列元件如SEQ ID NO：2中第6722-7310位核苷酸所示。
3’LTR序列元件如SEQ ID NO：2中第7382-7615位核苷酸所示。
bGH poly(A)signal序列元件如SEQ ID NO：2中第7647-7871位核苷酸所示。
质粒pKG-GE3具体如SEQ ID NO：2所示。
质粒pKG-U6gRNA中，具有SEQ ID NO：3中第2280-2637位核苷酸所示的DNA分子。
质粒pKG-U6gRNA具体如SEQ ID NO：3所示。
猪ADA基因信息：编码adenosine deaminase；位于17号染色体；GeneID为100625920，Sus scrofa。猪ADA基因编码的蛋白质如SEQ ID NO：4所示。基因组DNA中，猪ADA基因具有12个外显子，其中第4外显子及其上下游各500bp序列如SEQ ID NO：5所示。猪ADA基因为具有sgRNA ADA-g7的靶点的基因。猪ADA基因为具有SEQ ID NO：5所示区段的基因。
猪DQA基因信息：编码SLA class II histocompatibility antigen,DQ haplotype D alpha chain precurso；位于7号染色体；GeneID为100153387，Sus scrofa。猪DQA基因编码的蛋白质如SEQ ID NO：14所示(NCBI中的示例性序列)。基因组DNA中，猪DQA基因具有5个外显子，其中第2外显子及其上下游各500bp序列如SEQ ID NO：15所示(本发明中江香猪的测序结果)。猪DQA基因为具有sgRNA DQA-gn2的靶点的基因。猪DQA基因为具有SEQ ID NO：15所示区段的基因。
猪DRA基因信息：编码MHC class II DR-alpha precursor；位于7号染色体；GeneID为100135040，Sus scrofa。猪DRA基因编码的蛋白质如SEQ ID NO：26所示(NCBI中的示例性序列)。基因组DNA中，猪DRA基因具有5个外显子，其中第2外显子及其上下游各500bp序列如SEQ ID NO：27所示(本发明中江香猪的测序结果)。猪DRA基因为具有sgRNA DRA-g1的靶点的基因。猪DRA基因为具有SEQ ID NO：27所示区段的基因。
猪IL2RG基因信息：编码interleukin 2 receptor subunit gamma；位于X染色体；GeneID为397156，Sus scrofa。猪IL2RG基因编码的蛋白质如SEQ ID NO：32所示。基因组DNA中，猪IL2RG基因具有9个外显子，其中第4外显子及其上下游各500bp序列如SEQ ID NO：33所示。猪IL2RG基因为具有sgRNA IL2RG-g7的靶点的基因。猪DRA基因为具有SEQ ID NO：33所示区段的基因。
以上任一所述免疫缺陷具体可为重症免疫缺陷。
与现有技术相比，本发明至少具有如下有益效果：
(1)本发明研究对象(猪)比其他动物(大小鼠、灵长类)具有更好的应用性。目前未有任何大动物重症免疫缺陷模型被成功研发。大小鼠等啮齿类动物不论从体型、器官大小、生理、病理等方面都与人相差巨大，无法真实地模拟人类正常的生理、病理状态。研究表明，95％以上在大小鼠中验证有效的药物在人类临床试验中是无效的。就大动物而言，灵长类是与人亲缘关系最近的动物，但其体型小、性成熟晚(6-7岁开始交配)，且为单胎动物，群体扩繁速度极慢，饲养成本也很高。另外，灵长类动物克隆效率低、难度大、成本高。而猪作为模型动物就没有上述缺点，猪是除灵长类外与人亲缘关系最近的动物，其体型、体重、器官大小等与人相近，在解剖学、生理学、营养代谢、疾病发病机制等方面与人类极为相似。同时，猪的性成熟早(4-6个月)，繁殖力高，一窝多胎，在2-3年内即可形成一个较大群体。另外，猪的克隆技术非常成熟，克隆及饲养成本较灵长类低得多；而且猪作为人类长期以来的肉食性动物，用猪作为疾病模型动物在动物保护和伦理等方面的阻力相对较小。
(2)采用本发明改造的cas9高效表达载体进行基因编辑,编辑效率比原载体显著提高。
(3)采用本发明改造的Cas9高效表达载体进行基因编辑，通过靶标基因PCR产物测序结果可分析出所获细胞的基因型(纯合突变包括双等位基因相同突变和双等位基因不同突变、杂合突变或野生型)，获得纯合突变的概率为10％～20％；另外，利用所得到的纯合突变单克隆细胞株进行体细胞核移植可直接得到含靶标基因纯合突变的克隆猪，并且该纯合突变可稳定遗传。
附图说明
图1为质粒pX330的结构示意图。
图2为质粒pKG-GE3的结构示意图。
图3为质粒pKG-U6gRNA的结构示意图。
图4为将20bp左右的DNA分子(用于转录形成gRNA的靶序列结合区)插入质粒pKG-U6gRNA的示意图。
图5为实施例2的步骤三中MSTN三组的电泳图。
图6为实施例2的步骤三中FNDC5三组的电泳图。
图7为实施例3的步骤一中以8只猪的基因组DNA为模板采用ADA-GT-F259/ADA-GT-R1005组成的引物对进行PCR扩增后的电泳图。
图8为实施例3的步骤三中各种具有粘性末端的双链DNA分子。
图9为实施例3的步骤四中的测序峰图。
图10为实施例4的步骤一中以8只猪的基因组DNA为模板采用DQA-GT-F534/DQA-GT-R1332组成的引物对进行PCR扩增后的电泳图。
图11为实施例4的步骤三中各种具有粘性末端的双链DNA分子。
图12为实施例4的步骤四中的测序峰图。
图13为实施例5的步骤一中以8只猪的基因组DNA为模板采用DRA-GT-F326/DRA-GT-R1192组成的引物对进行PCR扩增后的电泳图。
图14为实施例5的步骤三中各种具有粘性末端的双链DNA分子。
图15为实施例5的步骤四中的测序峰图。
图16为实施例6的步骤一中以8只猪的基因组DNA为模板采用IL2RG-GT-F4543/IL2RG-GT-R5180组成的引物对进行PCR扩增后的电泳图。
图17为实施例6的步骤三中各种具有粘性末端的双链DNA分子。
图18为实施例6的步骤四中的测序峰图。
图19为实施例7中得到的单克隆细胞的靶基因PCR产物电泳图(采用ADA-nnF229和ADA-nnR456组成的引物对)。
图20为实施例7中得到的单克隆细胞的靶基因PCR产物电泳图(采用DQA-F643和DQA-R1022组成的引物对)。
图21为实施例7中得到的单克隆细胞的靶基因PCR产物电泳图(采用DRA-F573和DRA-R968组成的引物对)。
图22为实施例7中得到的单克隆细胞的靶基因PCR产物电泳图(采用IL2RG-nF33和IL2RG-nR460组成的引物对)。
图23为表1中部分单克隆细胞的靶基因测序峰图。
图24为表2中部分单克隆细胞的靶基因测序峰图。
图25为表3中部分单克隆细胞的靶基因测序峰图。
图26为表4中部分单克隆细胞的靶基因测序峰图。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。如无特殊说明，以下实施例中的定量试验，均设置三次重复实验，结果取平均值。完全培养液(％为体积比)：15％胎牛血清(Gibco)+83％DMEM培养基(Gibco)+1％Penicillin-Streptomycin(Gibco)+1％HEPES(Solarbio)。细胞培养条件：37℃，5％CO 2、5％O 2的恒温培养箱。
实施例3至6中的8只猪均为刚出生从江香猪，其中雌性4只(分别命名1、2、3、4)、雄性4只(分别命名为A、B、C、D)。
制备猪原代成纤维细胞的方法：①取猪耳组织0.5g，除毛，然后用75﹪酒精浸泡30-40s，然后用含5％(体积比)Penicillin-Streptomycin(Gibco)的PBS缓冲液洗涤5次，然后用PBS缓冲液洗涤一次；②用剪刀将组织剪碎，采用5mL 1％胶原酶溶液(Sigma)，37℃消化1h，然后500g离心5min，弃上清；③将沉淀用1mL完全培养液重悬，然后铺入含10mL完全培养基并已用0.2％明胶(VWR)封盘的直径为9cm的细胞培养皿中，培养至细胞长满皿底60％左右；④完成步骤③后，采用胰蛋白酶消化并收集细胞，使用细胞冻存液(90％完全培养基+10％DMSO，体积比)将细胞冻存。
用于实施例2至7中的猪原代成纤维细胞均获自上述命名为2的猪(雌性，血型AO)。
实施例1、质粒的制备
制备质粒pX330-U6-Chimeric_BB-CBh-hSpCas9，如SEQ ID NO：1所示。质粒pX330-U6-Chimeric_BB-CBh-hSpCas9，简称质粒pX330。
制备质粒pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO，如SEQ ID NO：2所示。质粒pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO，简称质粒pKG-GE3。
制备质粒pKG-U6gRNA，如SEQ ID NO：3所示。
质粒pX330、质粒pKG-GE3、质粒pKG-U6gRNA均为环形质粒。
质粒pX330的结构示意图见图1。SEQ ID NO：1中，第440-725位核苷酸组成CMV增强子，第727-1208位核苷酸组成chickenβ-actin启动子，第1304-1324位核苷酸编码SV40核定位信号(NLS)，第1325-5449位核苷酸编码Cas9蛋白，第5450-5497 位核苷酸编码nucleoplasmin核定位信号(NLS)。
质粒pKG-GE3的结构示意图见图2。SEQ ID NO：2中，第395-680位核苷酸组成CMV增强子，第682-890位核苷酸组成EF1a启动子，第986-1006位核苷酸编码核定位信号(NLS)，第1016-1036位核苷酸编码核定位信号(NLS)，第1037-5161位核苷酸编码Cas9蛋白，第5162-5209位核苷酸编码核定位信号(NLS)，第5219-5266位核苷酸编码核定位信号(NLS)，第5276-5332位核苷酸编码自剪切多肽P2A(自剪切多肽P2A的氨基酸序列为“ATNFSLLKQAGDVEENPGP”，发生自剪切的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间)，第5333-6046位核苷酸编码EGFP蛋白，第6056-6109位核苷酸编码自裂解多肽T2A(自裂解多肽T2A的氨基酸序列为“EGRGSLLTCGDVEENPGP”，发生自裂解的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间)，第6110-6703位核苷酸编码Puromycin蛋白(简称Puro蛋白)，第6722-7310位核苷酸组成WPRE序列元件，第7382-7615位核苷酸组成3’LTR序列元件，第7647-7871位核苷酸组成bGH poly(A)signal序列元件。SEQ ID NO：2中，第911-6706形成融合基因，表达融合蛋白。由于自剪切多肽P2A和自裂解多肽T2A的存在，融合蛋白自发形成如下三个蛋白：具有Cas9蛋白的蛋白、具有EGFP蛋白的蛋白和具有Puro蛋白的蛋白。
与质粒pX330相比，质粒pKG-GE3主要进行了如下改造：①去除残留的gRNA骨架序列(GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTT)，降低干扰；②将原有chickenβ-actin启动子改造为具更高表达活性的EF1a启动子，增加Cas9基因的蛋白表达能力；③在Cas9基因的上游和下游均增加核定位信号编码基因(NLS)，增加Cas9蛋白的核定位能力；④原质粒无任何真核细胞筛选标记，不利于阳性转化细胞的筛选和富集，依次在Cas9基因的下游插入P2A-EGFP-T2A-PURO编码基因，赋予载体荧光和真核细胞抗性筛选能力；⑤插入WPRE元件和3’LTR序列元件，增强Cas9基因的蛋白翻译能力。
质粒pKG-U6gRNA的结构示意图见图3。SEQ ID NO：3中，第2280-2539位核苷酸组成hU6启动子，第2558-2637位核苷酸用于转录形成gRNA骨架。使用时，将20bp左右的DNA分子(用于转录形成gRNA的靶序列结合区)插入质粒pKG-U6gRNA，形成重组质粒，示意图见图4,在细胞中重组质粒转录得到gRNA。
实施例2、质粒pX330和质粒pKG-GE3的效果比较
选择位于MSTN基因的两个gRNA靶点：
MSTN-gRNA1的靶点：5’-GCTGATTGTTGCTGGTCCCG-3’；
MSTN-gRNA2的靶点：5’-TTTCCAGGCGAAGTTTACTG-3’。
选择位于FNDC5基因的两个gRNA靶点：
FNDC5-gRNA1的靶点：5’-TGTACTCAGTGTCCTCCTCC-3’；
FNDC5-gRNA2的靶点：5’-GCTCTTCAAGACGCCTCGCG-3’。
用于扩增包含靶点的片段的引物为：
MSTN-F896：5’-TCTCTCAGACAGTGCAGGCATTA-3’；
MSTN-R1351：5’-CGTTTCCGTCGTAGCGTGATAAT-3’。
FNDC5-F209：5’-CAGTTCTCACTTGATGGCCTTGG-3’；
FNDC5-R718：5’-AGGGGTCTGGGGAGGAATGG-3’。
一、制备重组质粒
取质粒pKG-U6gRNA，用限制性内切酶BbsI进行酶切，回收载体骨架(约3kb的线性大片段)。
分别合成MSTN-1S和MSTN-1A，然后混合并进行退火，得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(MSTN-1)。
分别合成MSTN-2S和MSTN-2A，然后混合并进行退火，得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(MSTN-2)。
分别合成FNDC5-1S和FNDC5-1A，然后混合并进行退火，得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(FNDC5-1)。
分别合成FNDC5-2S和FNDC5-2A，然后混合并进行退火，得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(FNDC5-2)。
MSTN-1S：5’-caccGCTGATTGTTGCTGGTCCCG-3’；
MSTN-1A：5’-aaacCGGGACCAGCAACAATCAGC-3’。
MSTN-2S：5’-caccgTTTCCAGGCGAAGTTTACTG-3’；
MSTN-2A：5’-aaacCAGTAAACTTCGCCTGGAAAc-3’。
FNDC5-1S：5’-caccgTGTACTCAGTGTCCTCCTCC-3’；
FNDC5-1A：5’-aaacGGAGGAGGACACTGAGTACAc-3’。
FNDC5-2S：5’-caccGCTCTTCAAGACGCCTCGCG-3’；
FNDC5-2A：5’-aaacCGCGAGGCGTCTTGAAGAGC-3’。
二、质粒pX330和质粒pKG-GE3的效果比较
1、共转染
MSTN-B组：将质粒pKG-U6gRNA(MSTN-1)和质粒pKG-U6gRNA(MSTN-2)共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.46μg质粒pKG-U6gRNA(MSTN-1)：0.46μg质粒pKG-U6gRNA(MSTN-2)。
MSTN-330组：将质粒pKG-U6gRNA(MSTN-1)、质粒pKG-U6gRNA(MSTN-2)和质粒pX330共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.46μg质粒pKG-U6gRNA(MSTN-1)：0.46μg质粒pKG-U6gRNA(MSTN-2)：1.08μg质粒pX330。
MSTN-KG组：将质粒pKG-U6gRNA(MSTN-1)、质粒pKG-U6gRNA(MSTN-2)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.46 μg质粒pKG-U6gRNA(MSTN-1)：质粒0.46μg pKG-U6gRNA(MSTN-2)：1.08μg质粒pKG-GE3。
FNDC5-B组：将质粒pKG-U6gRNA(FNDC5-1)和质粒pKG-U6gRNA(FNDC5-2)共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.46μg质粒pKG-U6gRNA(FNDC5-1)：0.46μg质粒pKG-U6gRNA(FNDC5-2)。
FNDC5-330组：将质粒pKG-U6gRNA(FNDC5-1)、质粒pKG-U6gRNA(FNDC5-2)和质粒pX330共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.46μg质粒pKG-U6gRNA(FNDC5-1)：0.46μg质粒pKG-U6gRNA(FNDC5-2)：1.08μg质粒pX330。
FNDC5-KG组：将质粒pKG-U6gRNA(FNDC5-1)、质粒pKG-U6gRNA(FNDC5-2)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.46μg质粒pKG-U6gRNA(FNDC5-1)：0.46μg质粒pKG-U6gRNA(FNDC5-2)：1.08μg质粒pKG-GE3。
共转染采用电击转染的方式，采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为：1450V、10ms、3pulse)。
2、完成步骤1后，采用完全培养液培养16-18小时，然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后，采用胰蛋白酶消化并收集细胞，提取基因组DNA，采用MSTN-F896和MSTN-R1351组成的引物对(MSTN的三组)进行PCR扩增，或者采用FNDC5-F209和FNDC5-R718组成的引物对(FNDC5的三组)进行PCR扩增，然后进行电泳。
MSTN的三组的结果见图5。
FNDC5的三组的结果见图6。
MSTN-330组基因缺失突变效率为27.6％，MSTN-KG组基因缺失突变效率为86.5％。FNDC5-330组基因缺失突变效率为18.6％，FNDC5-KG组基因缺失突变效率为81.7％。结果表明，与采用质粒pX330相比，采用质粒pKG-GE3使得基因编辑效率显著提高。
实施例3、ADA基因敲除的靶点筛选
一、ADA基因敲除预设靶点及邻近基因组序列保守性分析
猪ADA基因信息：编码adenosine deaminase；位于17号染色体；GeneID为100625920，Sus scrofa。猪ADA基因编码的蛋白质如SEQ ID NO：4所示。基因组DNA中，猪ADA基因具有12个外显子，其中第4外显子及其上下游各500bp序列如SEQ ID NO：5所示。
分别以8只猪的基因组DNA为模板，采用引物ADA-GT-F259/ADA-GT-R1005组成的引物对进行PCR扩增，然后进行电泳，见图7。回收PCR扩增产物并进行测序，将测序结果与公共数据库中的ADA基因序列进行比对分析。根据比对结果，设计用于检 测突变的引物(引物本身避开可能的突变位点)。设计的用于检测突变的引物为：ADA-nnF229/ADA-nnR456。
ADA-GT-F259：5’-GTTAAGGATCTGGTGTTGCGGTG-3’；
ADA-GT-R1005：5’-GTTCACACTCCTAGACTCCAGCC-3’。
ADA-nnF229：5’-GAGGCCGTCAAAAGGATTGC-3’；
ADA-nnR456：5’-CAAAGTCTCTCTTGGGTCAGGG-3’。
二、筛选靶点
通过筛选NGG(避开可能的突变位点)初步筛选到若干靶点，经过预实验进一步从中筛选到8个靶点。
8个靶点分别如下：
sgRNA ADA-g1靶点：5’-AAGGATTGCCTACGAGTTTG-3’；
sgRNA ADA-g2靶点：5’-TTGGAGTTGGCCAGCAGGTG-3’；
sgRNA ADA-g3靶点：5’-TTTCATCTCCACAAACTCGT-3’；
sgRNA ADA-g4靶点：5’-TCAGCCTGGTTCCAGGGGAT-3’；
sgRNA ADA-g6靶点：5’-CCTGCTGGCCAACTCCAAAG-3’；
sgRNA ADA-g7靶点：5’-GGAGGGCGTGGTGTACGTGG-3’；
sgRNA ADA-g8靶点：5’-CAAGGAGGGCGTGGTGTACG-3’；
sgRNA ADA-g9靶点：5’-TGTGGAGATGAAAGCCAAGG-3’。
三、制备重组质粒
取质粒pKG-U6gRNA，用限制性内切酶BbsI进行酶切，回收载体骨架(约3kb的线性大片段)。
分别合成ADA-g1S和ADA-g1A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图8A)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(ADA-g1)。质粒pKG-U6gRNA(ADA-g1))表达SEQ ID NO：6所示的sgRNA ADA-g1。
SEQ ID NO：6：
Figure PCTCN2020124634-appb-000001
分别合成ADA-g2S和ADA-g2A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图8B)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(ADA-g2)。质粒pKG-U6gRNA(ADA-g2)表达SEQ ID NO：7所示的sgRNA ADA-g2。
SEQ ID NO：7：
Figure PCTCN2020124634-appb-000002
分别合成ADA-g3S和ADA-g3A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图8C)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(ADA-g3)。质粒pKG-U6gRNA(ADA-g3)表达SEQ ID NO：8所示的sgRNA ADA-g3。
SEQ ID NO：8：
Figure PCTCN2020124634-appb-000003
分别合成ADA-g4S和ADA-g4A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图8D)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(ADA-g4)。质粒pKG-U6gRNA(ADA-g4)表达SEQ ID NO：9所示的sgRNA ADA-g4。
SEQ ID NO：9：
Figure PCTCN2020124634-appb-000004
分别合成ADA-g6S和ADA-g6A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图8E)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(ADA-g6)。质粒pKG-U6gRNA(ADA-g6)表达SEQ ID NO：10所示的sgRNA ADA-g6。
SEQ ID NO：10：
Figure PCTCN2020124634-appb-000005
分别合成ADA-g7S和ADA-g7A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图8F)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(ADA-g7)。质粒pKG-U6gRNA(ADA-g7)表达SEQ ID NO：11所示的sgRNA ADA-g7。
SEQ ID NO：11：
Figure PCTCN2020124634-appb-000006
分别合成ADA-g8S和ADA-g8A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图8G)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(ADA-g8)。质粒pKG-U6gRNA(ADA-g8)表达SEQ ID NO：12所示的sgRNA ADA-g8。
SEQ ID NO：12：
Figure PCTCN2020124634-appb-000007
分别合成ADA-g9S和ADA-g9A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图8H)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(ADA-g9)。质粒pKG-U6gRNA(ADA-g9)表达SEQ ID NO：13所示的sgRNA ADA-g9。
SEQ ID NO：13：
Figure PCTCN2020124634-appb-000008
sgRNA-ADA-1S：5’-caccgAAGGATTGCCTACGAGTTTG-3’；
sgRNA-ADA-1A：5’-aaacCAAACTCGTAGGCAATCCTTc-3’。
sgRNA-ADA-2S：5’-caccgTTGGAGTTGGCCAGCAGGTG-3’；
sgRNA-ADA-2A：5’-aaacCACCTGCTGGCCAACTCCAAc-3’。
sgRNA-ADA-3S：5’-caccgTTTCATCTCCACAAACTCGT-3’；
sgRNA-ADA-3A：5’-aaacACGAGTTTGTGGAGATGAAAc-3’。
sgRNA-ADA-4S：5’-caccgTCAGCCTGGTTCCAGGGGAT-3’；
sgRNA-ADA-4A：5’-aaacATCCCCTGGAACCAGGCTGAc-3’。
sgRNA-ADA-6S：5’-caccgCCTGCTGGCCAACTCCAAAG-3’；
sgRNA-ADA-6A：5’-aaacCTTTGGAGTTGGCCAGCAGGc-3’。
sgRNA-ADA-7S：5’-caccGGAGGGCGTGGTGTACGTGG-3’；
sgRNA-ADA-7A：5’-aaacCCACGTACACCACGCCCTCC-3’。
sgRNA-ADA-8S：5’-caccgCAAGGAGGGCGTGGTGTACG-3’；
sgRNA-ADA-8A：5’-aaacCGTACACCACGCCCTCCTTGc-3’。
sgRNA-ADA-9S：5’-caccgTGTGGAGATGAAAGCCAAGG-3’；
sgRNA-ADA-9A：5’-aaacCCTTGGCTTTCATCTCCACAc-3’。
sgRNA-ADA-1S、sgRNA-ADA-1A、sgRNA-ADA-2S、sgRNA-ADA-2A、sgRNA-ADA-3S、sgRNA-ADA-3A、sgRNA-ADA-4S、sgRNA-ADA-4A、sgRNA-ADA-6S、sgRNA-ADA-6A、sgRNA-ADA-7S、sgRNA-ADA-7A、sgRNA-ADA-8S、sgRNA-ADA-8A、sgRNA-ADA-9S、sgRNA-ADA-9A均为单链DNA分子。
四、不同靶点的编辑效率比较
1、共转染
第一组：将质粒pKG-U6gRNA(ADA-g1)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(ADA-g1)：1.238μg质粒pKG-GE3。
第二组：将质粒pKG-U6gRNA(ADA-g2)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(ADA-g2)：1.238μg质粒pKG-GE3。
第三组：将质粒pKG-U6gRNA(ADA-g3)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(ADA-g3)：1.238μg质粒pKG-GE3。
第四组：将质粒pKG-U6gRNA(ADA-g4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(ADA-g4)：1.238μg质粒pKG-GE3。
第五组：将质粒pKG-U6gRNA(ADA-g6)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(ADA-g6)：1.238μg质粒pKG-GE3。
第六组：将质粒pKG-U6gRNA(ADA-g7)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(ADA-g7)：1.238μg质粒pKG-GE3。
第七组：将质粒pKG-U6gRNA(ADA-g8)和质粒pKG-GE3共转染猪原代成纤维细 胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(ADA-g8)：1.238μg质粒pKG-GE3。
第八组：将质粒pKG-U6gRNA(ADA-g9)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(ADA-g9)：1.238μg质粒pKG-GE3。
第九组：猪原代成纤维细胞，未进行任何转染操作。
共转染采用电击转染的方式，采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为：1450V、10ms、3pulse)。
2、完成步骤1后，采用完全培养液培养16-18小时，然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后，采用胰蛋白酶消化并收集细胞，提取基因组DNA，采用ADA-nnF229和ADA-nnR456组成的引物对进行PCR扩增，然后进行电泳并测序，结果见图9。
通过利用Synthego ICE工具分析测序峰图得出不同靶点的编辑效率。第一组至第九组不同靶点的编辑效率依次为19％、17％、9％、0％、2％、35％、29％、20％和0％。结果表明，第六组编辑效率最高，sgRNA ADA-g7为最优靶点。
实施例4、DQA基因敲除的靶点筛选
一、DQA基因敲除预设靶点及邻近基因组序列保守性分析
猪DQA基因信息：编码SLA class II histocompatibility antigen,DQ haplotype D alpha chain precurso；位于7号染色体；GeneID为100153387，Sus scrofa。猪DQA基因编码的蛋白质如SEQ ID NO：14所示(NCBI中的示例性序列)。基因组DNA中，猪DQA基因具有5个外显子，其中第2外显子及其上下游各500bp序列如SEQ ID NO：15所示(本发明中从江香猪的测序结果)。
分别以8只猪的基因组DNA为模板，采用引物DQA-GT-F534/DQA-GT-R1332组成的引物对进行PCR扩增，然后进行电泳，见图10。回收PCR扩增产物并进行测序，将测序结果与公共数据库中的DQA基因序列进行比对分析。根据比对结果，设计用于检测突变的引物(引物本身避开可能的突变位点)。设计的用于检测突变的引物为：DQA-F643/DQA-R1022。
DQA-GT-F534：5’-TTGCAAAGATAAGGAGGCTTCGC-3’；
DQA-GT-R1332：5’-AGCTCTTGTTTCCCTTCTGCTCA-3。
DQA-F643：5’-CAGATGAAGCCCTTGATATTTGA-3’；
DQA-R1022：5’-AGAAAGGCAGAATGATGAACACA-3’。
二、筛选靶点
通过筛选NGG(避开可能的突变位点)初步筛选到若干靶点，经过预实验进一步从中筛选到10个靶点。
10个靶点分别如下：
sgRNA DQA-g1靶点：5’-TTAAGCCATAGGAGGCAACA-3’；
sgRNA DQA-g2靶点：5’-GCCATAGGAGGCAACATGGT-3’；
sgRNA DQA-g3靶点：5’-CCATGAATTTGATGGCGACG-3’；
sgRNA DQA-g4靶点：5’-CCTCGTCGCCATCAAATTCA-3’；
sgRNA DQA-gn1靶点：5’-CTGGTAGACATTTAAGCCAT-3’；
sgRNA DQA-gn2靶点：5’-GTAGACATTTAAGCCATAGG-3’；
sgRNA DQA-gn3靶点：5’-TTAAATGTCTACCAGTCTTA-3’；
sgRNA DQA-gn4靶点：5’-AGACAGTCTCCTTCTTCCCC-3’；
sgRNA DQA-gn5靶点：5’-TGGGGAAGAAGGAGACTGTC-3’；
sgRNA DQA-gn6靶点：5’-TTGACCCACAGGGTGCACTG-3’。
三、制备重组质粒
取质粒pKG-U6gRNA，用限制性内切酶BbsI进行酶切，回收载体骨架(约3kb的线性大片段)。
分别合成DQA-g1S和DQA-g1A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图11A)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DQA-g1)。质粒pKG-U6gRNA(DQA-g1))表达SEQ ID NO：16所示的sgRNA DQA-g1。
SEQ ID NO：16：
Figure PCTCN2020124634-appb-000009
分别合成DQA-g2S和DQA-g2A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图11B)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DQA-g2)。质粒pKG-U6gRNA(DQA-g2)表达SEQ ID NO：17所示的sgRNA DQA-g2。
SEQ ID NO：17：
Figure PCTCN2020124634-appb-000010
分别合成DQA-g3S和DQA-g3A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图11C)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DQA-g3)。质粒pKG-U6gRNA(DQA-g3)表达SEQ ID NO：18所示的sgRNA DQA-g3。
SEQ ID NO：18：
Figure PCTCN2020124634-appb-000011
分别合成DQA-g4S和DQA-g4A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图11D)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DQA-g4)。质粒pKG-U6gRNA(DQA-g4)表达SEQ ID NO：19所示 的sgRNA DQA-g4。
SEQ ID NO：19：
Figure PCTCN2020124634-appb-000012
分别合成DQA-gn1S和DQA-gn1A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图11E)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DQA-gn1)。质粒pKG-U6gRNA(DQA-gn1)表达SEQ ID NO：20所示的sgRNA DQA-gn1。
SEQ ID NO：20：
Figure PCTCN2020124634-appb-000013
分别合成DQA-gn2S和DQA-gn2A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图11F)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DQA-gn2)。质粒pKG-U6gRNA(DQA-gn2)表达SEQ ID NO：21所示的sgRNA DQA-gn2。
SEQ ID NO：21：
Figure PCTCN2020124634-appb-000014
分别合成DQA-gn3S和DQA-gn3A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图11G)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DQA-gn3)。质粒pKG-U6gRNA(DQA-gn3)表达SEQ ID NO：22所示的sgRNA DQA-gn3。
SEQ ID NO：22：
Figure PCTCN2020124634-appb-000015
分别合成DQA-gn4S和DQA-gn4A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图11H)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DQA-gn4)。质粒pKG-U6gRNA(DQA-gn4)表达SEQ ID NO：23所示的sgRNA DQA-gn4。
SEQ ID NO：23：
Figure PCTCN2020124634-appb-000016
分别合成DQA-gn5S和DQA-gn5A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图11I)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DQA-gn5)。质粒pKG-U6gRNA(DQA-gn5)表达SEQ ID NO：24所示的sgRNA DQA-gn5。
SEQ ID NO：24：
Figure PCTCN2020124634-appb-000017
分别合成DQA-gn6S和DQA-gn6A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图11J)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DQA-gn6)。质粒pKG-U6gRNA(DQA-gn6)表达SEQ ID NO：25所示的sgRNA DQA-gn6。
SEQ ID NO：25：
Figure PCTCN2020124634-appb-000018
sgRNA-DQA-1S：5’-caccgTTAAGCCATAGGAGGCAACA-3’；
sgRNA-DQA-1A：5’-aaacTGTTGCCTCCTATGGCTTAAc-3’。
sgRNA-DQA-2S：5’-caccGCCATAGGAGGCAACATGGT-3’；
sgRNA-DQA-2A：5’-aaacACCATGTTGCCTCCTATGGC-3’。
sgRNA-DQA-3S：5’-caccgCCATGAATTTGATGGCGACG-3’；
sgRNA-DQA-3A：5’-aaacCGTCGCCATCAAATTCATGGc-3’。
sgRNA-DQA-4S：5’-caccgCCTCGTCGCCATCAAATTCA-3’；
sgRNA-DQA-4A：5’-aaacTGAATTTGATGGCGACGAGGc-3’。
sgRNA-DQA-n1S：5’-caccgCTGGTAGACATTTAAGCCAT-3’；
sgRNA-DQA-n1A：5’-aaacATGGCTTAAATGTCTACCAGc-3’。
sgRNA-DQA-n2S：5’-caccGTAGACATTTAAGCCATAGG-3’；
sgRNA-DQA-n2A：5’-aaacCCTATGGCTTAAATGTCTAC-3’。
sgRNA-DQA-n3S：5’-caccgTTAAATGTCTACCAGTCTTA-3’；
sgRNA-DQA-n3A：5’-aaacTAAGACTGGTAGACATTTAAc-3’。
sgRNA-DQA-n4S：5’-caccgAGACAGTCTCCTTCTTCCCC-3’；
sgRNA-DQA-n4A：5’-aaacGGGGAAGAAGGAGACTGTCTc-3’。
sgRNA-DQA-n5S：5’-caccgTGGGGAAGAAGGAGACTGTC-3’；
sgRNA-DQA-n5A：5’-aaacGACAGTCTCCTTCTTCCCCAc-3’。
sgRNA-DQA-n6S：5’-caccgTTGACCCACAGGGTGCACTG-3’；
sgRNA-DQA-n6A：5’-aaacCAGTGCACCCTGTGGGTCAAc-3’。
sgRNA-DQA-1S、sgRNA-DQA-1A、sgRNA-DQA-2S、sgRNA-DQA-2A、sgRNA-DQA-3S、sgRNA-DQA-3A、sgRNA-DQA-4S、sgRNA-DQA-4A、sgRNA-DQA-n1S、sgRNA-DQA-n1A、sgRNA-DQA-n2S、sgRNA-DQA-n2A、sgRNA-DQA-n3S、sgRNA-DQA-n3A、sgRNA-DQA-n4S、sgRNA-DQA-n4A、sgRNA-DQA-n5S、sgRNA-DQA-n5A、sgRNA-DQA-n6S、sgRNA-DQA-n6A均为单链DNA分子。
四、不同靶点的编辑效率比较
1、共转染
第一组：将质粒pKG-U6gRNA(DQA-g1)和质粒pKG-GE3共转染猪原代成纤维细 胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DQA-g1)：1.238μg质粒pKG-GE3。
第二组：将质粒pKG-U6gRNA(DQA-g2)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DQA-g2)：1.238μg质粒pKG-GE3。
第三组：将质粒pKG-U6gRNA(DQA-g3)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DQA-g3)：1.238μg质粒pKG-GE3。
第四组：将质粒pKG-U6gRNA(DQA-g4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DQA-g4)：1.238μg质粒pKG-GE3。
第五组：将质粒pKG-U6gRNA(DQA-gn1)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DQA-gn1)：1.238μg质粒pKG-GE3。
第六组：将质粒pKG-U6gRNA(DQA-gn2)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DQA-gn2)：1.238μg质粒pKG-GE3。
第七组：将质粒pKG-U6gRNA(DQA-gn3)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DQA-gn3)：1.238μg质粒pKG-GE3。
第八组：将质粒pKG-U6gRNA(DQA-gn4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DQA-gn4)：1.238μg质粒pKG-GE3。
第九组：将质粒pKG-U6gRNA(DQA-gn5)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DQA-gn5)：1.238μg质粒pKG-GE3。
第十组：将质粒pKG-U6gRNA(DQA-gn6)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DQA-gn6)：1.238μg质粒pKG-GE3。
第十一组：猪原代成纤维细胞，未进行任何转染操作。
共转染采用电击转染的方式，采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为：1450V、10ms、3pulse)。
2、完成步骤1后，采用完全培养液培养16-18小时，然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后，采用胰蛋白酶消化并收集细胞，提取基因组DNA，采用DQA-F643和DQA-R1022组成的引物对进行PCR扩增，然后进行电泳并测序，结果见 图12。
通过利用Synthego ICE工具分析测序峰图得出不同靶点的编辑效率。第一组至第十一组不同靶点的编辑效率依次为35％、29％、11％、0％、12％、44％、11％、0％、12％、40％和0％。结果表明，第六组编辑效率最高，sgRNA DQA-gn2为最优靶点。
实施例5、DRA基因敲除的靶点筛选
一、DRA基因敲除预设靶点及邻近基因组序列保守性分析
猪DRA基因信息：编码MHC class II DR-alpha precursor；位于7号染色体；GeneID为100135040，Sus scrofa。猪DRA基因编码的蛋白质如SEQ ID NO：26所示(NCBI中的示例性序列)。基因组DNA中，猪DRA基因具有5个外显子，其中第2外显子及其上下游各500bp序列如SEQ ID NO：27所示(本发明中从江香猪的测序结果)。
分别以8只猪的基因组DNA为模板，采用引物DRA-GT-F326/DRA-GT-R1192组成的引物对进行PCR扩增，然后进行电泳，见图13。回收PCR扩增产物并进行测序，将测序结果与公共数据库中的DRA基因序列进行比对分析。根据比对结果，设计用于检测突变的引物(引物本身避开可能的突变位点)。设计的用于检测突变的引物为：DRA-F573/DRA-R968。
DRA-GT-F326：5’-TTTCACGGACAGTCACATGGAGT-3’；
DRA-GT-R1192：5’-ATACCTAGCTCTGAAATCCGCCC-3’。
DRA-F573：5’-TCATCGCCTTCTCTATTTTCCAC-3’；
DRA-R968：5’-CCCCTGGAAGGAAAAGTAAGTCA-3’。
二、筛选靶点
通过筛选NGG(避开可能的突变位点)初步筛选到若干靶点，经过预实验进一步从中筛选到4个靶点。
4个靶点分别如下：
sgRNA DRA-g1靶点：5’-TCCACGTGGATATGGAAAAG-3’；
sgRNA DRA-g2靶点：5’-CCCTCTTTTCCATATCCACG-3’；
sgRNA DRA-g3靶点：5’-AGCTGTGGACAAAGCCAACC-3’；
sgRNA DRA-g4靶点：5’-TGCACCCTGAGCCTCAAAGC-3’。
三、制备重组质粒
取质粒pKG-U6gRNA，用限制性内切酶BbsI进行酶切，回收载体骨架(约3kb的线性大片段)。
分别合成DRA-g1S和DRA-g1A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图14A)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DRA-g1)。质粒pKG-U6gRNA(DRA-g1)表达SEQ ID NO：28所示的sgRNA DRA-g1。
SEQ ID NO：28：
Figure PCTCN2020124634-appb-000019
分别合成DRA-g2S和DRA-g2A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图14B)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DRA-g2)。质粒pKG-U6gRNA(DRA-g2)表达SEQ ID NO：29所示的sgRNA DRA-g2。
SEQ ID NO：29：
Figure PCTCN2020124634-appb-000020
分别合成DRA-g3S和DRA-g3A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图14C)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DRA-g3)。质粒pKG-U6gRNA(DRA-g3)表达SEQ ID NO：30所示的sgRNA DRA-g3。
SEQ ID NO：30：
Figure PCTCN2020124634-appb-000021
分别合成DRA-g4S和DRA-g4A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图14D)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(DRA-g4)。质粒pKG-U6gRNA(DRA-g4)表达SEQ ID NO：31所示的sgRNA DRA-g4。
SEQ ID NO：31：
Figure PCTCN2020124634-appb-000022
sgRNA-DRA-1S：5’-caccgTCCACGTGGATATGGAAAAG-3’；
sgRNA-DRA-1A：5’-aaacCTTTTCCATATCCACGTGGAc-3’。
sgRNA-DRA-2S：5’-caccgCCCTCTTTTCCATATCCACG-3’；
sgRNA-DRA-2A：5’-aaacCGTGGATATGGAAAAGAGGGc-3’。
sgRNA-DRA-3S：5’-caccgAGCTGTGGACAAAGCCAACC-3’；
sgRNA-DRA-3A：5’-aaacGGTTGGCTTTGTCCACAGCTc-3’。
sgRNA-DRA-4S：5’-caccgTGCACCCTGAGCCTCAAAGC-3’；
sgRNA-DRA-4A：5’-aaacGCTTTGAGGCTCAGGGTGCAc-3’。
sgRNA-DRA-1S、sgRNA-DRA-1A、sgRNA-DRA-2S、sgRNA-DRA-2A、sgRNA-DRA-3S、sgRNA-DRA-3A、sgRNA-DRA-4S、sgRNA-DRA-4A均为单链DNA分子。
四、不同靶点的编辑效率比较
1、共转染
第一组：将质粒pKG-U6gRNA(DRA-g1)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DRA-g1)：1.238μg质粒pKG-GE3。
第二组：将质粒pKG-U6gRNA(DRA-g2)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DRA-g2)：1.238μg质粒pKG-GE3。
第三组：将质粒pKG-U6gRNA(DRA-g3)和质粒pKG-GE3共转染猪原代成纤维细 胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DRA-g3)：1.238μg质粒pKG-GE3。
第四组：将质粒pKG-U6gRNA(DRA-g4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(DRA-g4)：1.238μg质粒pKG-GE3。
第五组：猪原代成纤维细胞，未进行任何转染操作。
共转染采用电击转染的方式，采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为：1450V、10ms、3pulse)。
2、完成步骤1后，采用完全培养液培养16-18小时，然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后，采用胰蛋白酶消化并收集细胞，提取基因组DNA，采用DRA-F573和DRA-R968组成的引物对进行PCR扩增，然后进行电泳并测序，结果见图15。
通过利用Synthego ICE工具分析测序峰图得出不同靶点的编辑效率。第一组至第五组不同靶点的编辑效率依次为52％、45％、24％、20％和0％。结果表明，第一组编辑效率最高，sgRNA DRA-g1为最优靶点。
实施例6、IL2RG基因敲除的靶点筛选
一、IL2RG基因敲除预设靶点及邻近基因组序列保守性分析
猪IL2RG基因信息：编码interleukin 2receptor subunit gamma；位于X染色体；
GeneID为397156，Sus scrofa。猪IL2RG基因编码的蛋白质如SEQ ID NO：32所示。基因组DNA中，猪IL2RG基因具有9个外显子，其中第4外显子及其上下游各500bp序列如SEQ ID NO：33所示。
分别以8只猪的基因组DNA为模板，采用引物IL2RG-GT-F4543/IL2RG-GT-R5180组成的引物对进行PCR扩增，然后进行电泳，见图16。回收PCR扩增产物并进行测序，将测序结果与公共数据库中的IL2RG基因序列进行比对分析。根据比对结果，设计用于检测突变的引物(引物本身避开可能的突变位点)。设计的用于检测突变的引物为：IL2RG-nF33/IL2RG-nR460。
IL2RG-GT-F4543：5’-ATATAGCACAGGGGAGGGAGGAA-3’；
IL2RG-GT-R5180：5’-AGGGTGCGAAGGGTCAGATTC-3’；
IL2RG-nF33：5’-CCCAGGCTTCCCACTATATTCTC-3’；
IL2RG-nR460：5’-CCATTGGATCCCTCACTTCTTCT-3’。
二、筛选靶点
通过筛选NGG(避开可能的突变位点)初步筛选到若干靶点，经过预实验进一步从中筛选到9个靶点。
9个靶点分别如下：
sgRNA IL2RG-g1靶点：5’-CCTGTAGTTTTAGCGTCTGT-3’；
sgRNA IL2RG-g2靶点：5’-CAACAAATGTTTGGTAGAGG-3’；
sgRNA IL2RG-g3靶点：5’-GATGATAAAGTCCAGGAGTG-3’；
sgRNA IL2RG-g4靶点：5’-CTGGACTTTATCATCATTAG-3’；
sgRNA IL2RG-g5靶点：5’-TTGTCCAGCTCCAGGACCCA-3’；
sgRNA IL2RG-g6靶点：5’-GGCCACTATCTATTCTCTGA-3’；
sgRNA IL2RG-g7靶点：5’-TCCCTTCAGAGAATAGATAG-3’；
sgRNA IL2RG-g8靶点：5’-AACATTTGTTGTCCAGCTCC-3’；
sgRNA IL2RG-g9靶点：5’-TGTCCAGCTCCAGGACCCAC-3’。
三、制备重组质粒
取质粒pKG-U6gRNA，用限制性内切酶BbsI进行酶切，回收载体骨架(约3kb的线性大片段)。
分别合成IL2RG-g1S和IL2RG-g1A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图17A)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(IL2RG-g1)。质粒pKG-U6gRNA(IL2RG-g1)表达SEQ ID NO：34所示的sgRNA IL2RG-g1。
SEQ ID NO：34：
Figure PCTCN2020124634-appb-000023
分别合成IL2RG-g2S和IL2RG-g2A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图17B)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(IL2RG-g2)。质粒pKG-U6gRNA(IL2RG-g2)表达SEQ ID NO：35所示的sgRNA IL2RG-g2。
SEQ ID NO：35：
Figure PCTCN2020124634-appb-000024
分别合成IL2RG-g3S和IL2RG-g3A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图17C)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(IL2RG-g3)。质粒pKG-U6gRNA(IL2RG-g3)表达SEQ ID NO：36所示的sgRNA IL2RG-g3。
SEQ ID NO：36：
Figure PCTCN2020124634-appb-000025
分别合成IL2RG-g4S和IL2RG-g4A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图17D)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(IL2RG-g4)。质粒pKG-U6gRNA(IL2RG-g4)表达SEQ ID NO：37所示的sgRNA IL2RG-g4。
SEQ ID NO：37：
Figure PCTCN2020124634-appb-000026
分别合成IL2RG-g5S和IL2RG-g5A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图17E)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(IL2RG-g5)。质粒pKG-U6gRNA(IL2RG-g5)表达SEQ ID NO：38所示的sgRNA IL2RG-g5。
SEQ ID NO：38：
Figure PCTCN2020124634-appb-000027
分别合成IL2RG-g6S和IL2RG-g6A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图17F)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(IL2RG-g6)。质粒pKG-U6gRNA(IL2RG-g6)表达SEQ ID NO：39所示的sgRNA IL2RG-g6。
SEQ ID NO：39：
Figure PCTCN2020124634-appb-000028
分别合成IL2RG-g7S和IL2RG-g7A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图17G)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(IL2RG-g7)。质粒pKG-U6gRNA(IL2RG-g7)表达SEQ ID NO：40所示的sgRNA IL2RG-g7。
SEQ ID NO：40：
Figure PCTCN2020124634-appb-000029
分别合成IL2RG-g8S和IL2RG-g8A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图17H)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(IL2RG-g8)。质粒pKG-U6gRNA(IL2RG-g8)表达SEQ ID NO：41所示的sgRNA IL2RG-g8。
SEQ ID NO：41：
Figure PCTCN2020124634-appb-000030
分别合成IL2RG-g9S和IL2RG-g9A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图17I)。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(IL2RG-g9)。质粒pKG-U6gRNA(IL2RG-g9)表达SEQ ID NO：42所示的sgRNA IL2RG-g9。
SEQ ID NO：42：
Figure PCTCN2020124634-appb-000031
sgRNA-IL2RG-1S：5’-caccgCCTGTAGTTTTAGCGTCTGT-3’；
sgRNA-IL2RG-1A：5’-aaacACAGACGCTAAAACTACAGGc-3’。
sgRNA-IL2RG-2S：5’-caccgCAACAAATGTTTGGTAGAGG-3’；
sgRNA-IL2RG-2A：5’-aaacCCTCTACCAAACATTTGTTGc-3’。
sgRNA-IL2RG-3S：5’-caccGATGATAAAGTCCAGGAGTG-3’；
sgRNA-IL2RG-3A：5’-aaacCACTCCTGGACTTTATCATC-3’。
sgRNA-IL2RG-4S：5’-caccgCTGGACTTTATCATCATTAG-3’；
sgRNA-IL2RG-4A：5’-aaacCTAATGATGATAAAGTCCAGc-3’。
sgRNA-IL2RG-5S：5’-caccgTTGTCCAGCTCCAGGACCCA-3’；
sgRNA-IL2RG-5A：5’-aaacTGGGTCCTGGAGCTGGACAAc-3’。
sgRNA-IL2RG-6S：5’-caccgGGCCACTATCTATTCTCTGA-3’；
sgRNA-IL2RG-6A：5’-aaacTCAGAGAATAGATAGTGGCCc-3’。
sgRNA-IL2RG-7S：5’-caccgTCCCTTCAGAGAATAGATAG-3’；
sgRNA-IL2RG-7A：5’-aaacCTATCTATTCTCTGAAGGGAc-3’。
sgRNA-IL2RG-8S：5’-caccgAACATTTGTTGTCCAGCTCC-3’；
sgRNA-IL2RG-8A：5’-aaacGGAGCTGGACAACAAATGTTc-3’。
sgRNA-IL2RG-9S：5’-caccgTGTCCAGCTCCAGGACCCAC-3’；
sgRNA-IL2RG-9A：5’-aaacGTGGGTCCTGGAGCTGGACAc-3’。
sgRNA-IL2RG-1S、sgRNA-IL2RG-1A、sgRNA-IL2RG-2S、sgRNA-IL2RG-2A、sgRNA-IL2RG-3S、sgRNA-IL2RG-3A、sgRNA-IL2RG-4S、sgRNA-IL2RG-4A、sgRNA-IL2RG-5S、sgRNA-IL2RG-5A、sgRNA-IL2RG-6S、sgRNA-IL2RG-6A、sgRNA-IL2RG-7S、sgRNA-IL2RG-7A、sgRNA-IL2RG-8S、sgRNA-IL2RG-8A、sgRNA-IL2RG-9S、sgRNA-IL2RG-9A均为单链DNA分子。
四、不同靶点的编辑效率比较
1、共转染
第一组：将质粒pKG-U6gRNA(IL2RG-g1)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(IL2RG-g1)：1.238μg质粒pKG-GE3。
第二组：将质粒pKG-U6gRNA(IL2RG-g2)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(IL2RG-g2)：1.238μg质粒pKG-GE3。
第三组：将质粒pKG-U6gRNA(IL2RG-g3)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(IL2RG-g3)：1.238μg质粒pKG-GE3。
第四组：将质粒pKG-U6gRNA(IL2RG-g4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(IL2RG-g4)：1.238μg质粒pKG-GE3。
第五组：将质粒pKG-U6gRNA(IL2RG-g5)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(IL2RG-g5)：1.238μg质粒pKG-GE3。
第六组：将质粒pKG-U6gRNA(IL2RG-g6)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(IL2RG-g6)：1.238μg质粒pKG-GE3。
第七组：将质粒pKG-U6gRNA(IL2RG-g7)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(IL2RG-g7)：1.238μg质粒pKG-GE3。
第八组：将质粒pKG-U6gRNA(IL2RG-g8)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(IL2RG-g8)：1.238μg质粒pKG-GE3。
第九组：将质粒pKG-U6gRNA(IL2RG-g9)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.762μg质粒pKG-U6gRNA(IL2RG-g9)：1.238μg质粒pKG-GE3。
第十组：猪原代成纤维细胞，未进行任何转染操作。
共转染采用电击转染的方式，采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为：1450V、10ms、3pulse)。
2、完成步骤1后，采用完全培养液培养16-18小时，然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后，采用胰蛋白酶消化并收集细胞，提取基因组DNA，采用IL2RG-nF33和IL2RG-nR460组成的引物对进行PCR扩增，然后进行电泳并测序，结果见图18。
通过利用Synthego ICE工具分析测序峰图得出不同靶点的编辑效率。第一组至第十组不同靶点的编辑效率依次为1％、0％、3％、5％、0％、46％、65％、18％、34％和0％。结果表明，第七组编辑效率最高，sgRNA IL2RG-g7为最优靶点。
实施例7、制备ADA、DQA、DRA和IL2RG基因编辑SCID单克隆细胞
1、共转染
将质粒pKG-U6gRNA(ADA-g7)、质粒pKG-U6gRNA(DQA-gn2)、质粒pKG-U6gRNA(DRA-g1)、质粒pKG-U6gRNA(IL2RG-g7)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.34μg质粒pKG-U6gRNA(ADA-g7)：0.34μg质粒pKG-U6gRNA(DQA-gn2)：0.34μg质粒pKG-U6gRNA(DRA-g1)：0.34μg质粒pKG-U6gRNA(IL2RG-g7)：1.64μg质粒pKG-GE3。
共转染采用电击转染的方式，采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为：1450V、10ms、3pulse)。
2、完成步骤1后，采用完全培养液培养16-18小时，然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后，采用胰蛋白酶消化并收集细胞，然后用完全培养液洗涤，然后用完全培养液重悬，然后分别挑取各个单克隆转移到96孔板中(每个孔1个细胞，每个孔中装有200μl完全培养液)，培养2周(每2-3天更换新的完全培养液)。
4、完成步骤3后，采用胰蛋白酶消化并收集细胞(每孔得到的细胞，约2/3接种到装有完全培养液的6孔板中，剩余的1/3收集在1.5mL离心管中)。
5、取步骤4的6孔板，培养直至细胞长至50％丰满度，采用胰蛋白酶消化并收集细胞，使用细胞冻存液(90％完全培养基+10％DMSO，体积比)将细胞冻存。
6、取步骤4的离心管，取细胞，提取基因组DNA，进行PCR扩增(分别采用ADA-nnF229和ADA-nnR456组成的引物对、DQA-F643和DQA-R1022组成的引物对、DRA-F573和DRA-R968组成的引物对、IL2RG-nF33和IL2RG-nR460组成的引物对)，然后进行电泳。将猪原代成纤维细胞作为野生型对照。
采用ADA-nnF229和ADA-nnR456组成的引物对的电泳图见图19。
采用DQA-F643和DQA-R1022组成的引物对的电泳图见图20。
采用DRA-F573和DRA-R968组成的引物对的电泳图见图21。
采用IL2RG-nF33和IL2RG-nR460组成的引物对的电泳图见图22。
7、完成步骤6后，回收PCR扩增产物并测序。
猪原代成纤维细胞的测序结果只有一种，其基因型为纯合野生型。如果某一单克隆细胞的测序结果有两种，一种与猪原代成纤维细胞的测序结果一致，另一种与猪原代成纤维细胞的测序结果相比发生了突变(突变包括一个或多个核苷酸的缺失、插入或替换)，该单克隆细胞的基因型为杂合型；如果某一单克隆细胞的测序结果为两种，均与猪原代成纤维细胞的测序结果相比发生了突变(突变包括一个或多个核苷酸的缺失、插入或替换)，该单克隆细胞的基因型为双等位基因不同突变型；如果某一单克隆细胞的测序结果为一种，且与猪原代成纤维细胞的测序结果相比发生了突变(突变包括一个或多个核苷酸的缺失、插入或替换)，该单克隆细胞的基因型为双等位基因相同突变型；如果某一单克隆细胞的测序结果为一种，且与猪原代成纤维细胞的测序结果一致，该单克隆细胞的基因型为纯合野生型。
ADA基因的编辑结果见表1。编号为31、43的单克隆细胞的基因型为双等位基因相同突变型。编号为45、52的单克隆细胞的基因型为双等位基因不同突变型。编号为39的单克隆细胞的基因型为杂合型。编号为4、6、12、14、20、28、29、34、48、50的单克隆细胞均显示为复杂的套峰，因此未能获得有效的序列，不能确定基因型和具体形式，但可以判断发生了基因编辑。得到的基因编辑单克隆细胞的比率为15/73。示例性的ADA的测序峰图见图23。
表1
Figure PCTCN2020124634-appb-000032
Figure PCTCN2020124634-appb-000033
Figure PCTCN2020124634-appb-000034
DQA基因的编辑结果见表2。编号为29、31、32、45、51、69的单克隆细胞的基因型为双等位基因相同突变型。编号为39、52、61的单克隆细胞的基因型为双等位基因不同突变型。编号为44、70、72的单克隆细胞的基因型为杂合型。编号 为4、6、12、14、20、28、34、46、67的单克隆细胞均显示为复杂的套峰，因此未能获得有效的序列，不能确定基因型和具体形式，但可以判断发生了基因编辑。得到的DQA基因编辑单克隆细胞的比率为21/71。示例性的DQA的测序峰图见图24。
表2
Figure PCTCN2020124634-appb-000035
Figure PCTCN2020124634-appb-000036
Figure PCTCN2020124634-appb-000037
DRA基因的编辑结果见表3。编号为31、50的单克隆细胞的基因型为双等位基因相同突变型。编号为69的单克隆细胞的基因型为杂合型。编号为6、12、29、45、52的单克隆细胞均显示为复杂的套峰，因此未能获得有效的序列，不能确定基因型和具体形式，但可以判断发生了基因编辑。DRA基因编辑单克隆细胞的比率为8/71。示例性的DRA的测序峰图见图25。
表3
Figure PCTCN2020124634-appb-000038
Figure PCTCN2020124634-appb-000039
Figure PCTCN2020124634-appb-000040
IL2RG基因的编辑结果见表4。编号为12、61的单克隆细胞的基因型为双等位基因相同突变型。编号为43、45、50的单克隆细胞的基因型为双等位基因不同突变型。编号为46、48、51、69、70的单克隆细胞的基因型为杂合型。编号为6、20、28、29、31、32、52的单克隆细胞均显示为复杂的套峰，因此未能获得有效的序列，不能确定基因型和具体形式，但可以判断发生了基因编辑。得到的IL2RG基因编辑单克隆细胞的比率为17/72。示例性的IL2RG的测序峰图见图26。
表4
Figure PCTCN2020124634-appb-000041
Figure PCTCN2020124634-appb-000042
Figure PCTCN2020124634-appb-000043
8、完成步骤7后，挑选IL2RG、ADA、DQA和DRA基因同时敲除的单克隆细胞。
通过分析，编号为6、12、29、31、45、52的单克隆细胞为ADA、DQA、DRA和IL2RG基因同时敲除的单克隆细胞。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明可用于通过基因编辑手段获得重症免疫缺陷猪模型，用于进行药物筛选、药效检测、疾病病理、基因治疗及细胞治疗等研究，能够为进一步的临床应用提供有效的实验数据，也为今后治愈人类重症免疫缺陷提供了有力的实验手段。本发明为重症免疫缺陷猪模型的制备奠定了坚实的基础，对于重症免疫缺陷药物的研发具有重大应用价值。

Claims (11)

  1. 一种制备重组细胞的方法，包括如下步骤：将质粒pKG-T6gRNA(ADA-g7)、质粒pKG-T6gRNA(DQA-gn2)、质粒pKG-T6gRNA(DRA-g1)、质粒pKG-T6gRNA(IL2RG-g7)和质粒pKG-GE3共转染猪细胞，得到ADA基因、DQA基因、DRA基因和IL2RG基因均发生突变的重组细胞；
    所述质粒pKG-T6gRNA(ADA-g7)转录得到sgRNA ADA-g7；
    所述质粒pKG-T6gRNA(DQA-gn2)转录得到sgRNA DQA-gn2；
    所述质粒pKG-T6gRNA(DRA-g1)转录得到sgRNA DRA-g1；
    所述质粒pKG-T6gRNA(IL2RG-g7)转录得到sgRNA IL2RG-g7；
    所述sgRNA ADA-g7的靶序列结合区如SEQ ID NO：11中第1-20位核苷酸所示；
    所述sgRNA DQA-gn2的靶序列结合区如SEQ ID NO：21中第1-20位核苷酸所示；
    所述sgRNA DRA-g1的靶序列结合区如SEQ ID NO：28中第1-20位核苷酸所示；
    所述sgRNA IL2RG-g7的靶序列结合区如SEQ ID NO：40中第1-20位核苷酸所示；
    质粒pKG-GE3中，具有特异融合基因；所述特异融合基因编码特异融合蛋白；
    所述特异融合蛋白自N端至C端依次包括如下元件：两个核定位信号、Cas9蛋白、两个核定位信号、自剪切多肽P2A、荧光报告蛋白、自裂解多肽T2A、抗性筛选标记蛋白；
    质粒pKG-GE3中，由EF1a启动子启动所述特异融合基因的表达；
    质粒pKG-GE3中，所述特异融合基因下游具有WPRE序列元件、3’LTR序列元件和bGH poly(A)signal序列元件。
  2. 权利要求1所述方法制备得到的重组细胞。
  3. 权利要求2所述重组细胞在制备免疫缺陷动物模型中的应用。
  4. 权利要求2所述重组细胞在制备免疫缺陷动物细胞模型中的应用。
  5. sgRNA组合，由sgRNA ADA-g7、sgRNA DQA-gn2、sgRNA DRA-g1和sgRNA IL2RG-g7组成；
    所述sgRNA ADA-g7的靶序列结合区如SEQ ID NO：11中第1-20位核苷酸所示；
    所述sgRNA DQA-gn2的靶序列结合区如SEQ ID NO：21中第1-20位核苷酸所示；
    所述sgRNA DRA-g1的靶序列结合区如SEQ ID NO：28中第1-20位核苷酸所示；
    所述sgRNA IL2RG-g7的靶序列结合区如SEQ ID NO：40中第1-20位核苷酸所示。
  6. 质粒组合，由质粒pKG-T6gRNA(ADA-g7)、质粒pKG-T6gRNA(DQA-gn2)、质粒pKG-T6gRNA(DRA-g1)和质粒pKG-T6gRNA(IL2RG-g7)组成；
    所述质粒pKG-T6gRNA(ADA-g7)转录得到sgRNA ADA-g7。；
    所述质粒pKG-T6gRNA(DQA-gn2)转录得到sgRNA DQA-gn2；
    所述质粒pKG-T6gRNA(DRA-g1)转录得到sgRNA DRA-g1；
    所述质粒pKG-T6gRNA(IL2RG-g7)转录得到sgRNA IL2RG-g7；
    所述sgRNA ADA-g7的靶序列结合区如SEQ ID NO：11中第1-20位核苷酸所示；
    所述sgRNA DQA-gn2的靶序列结合区如SEQ ID NO：21中第1-20位核苷酸所示；
    所述sgRNA DRA-g1的靶序列结合区如SEQ ID NO：28中第1-20位核苷酸所示；
    所述sgRNA IL2RG-g7的靶序列结合区如SEQ ID NO：40中第1-20位核苷酸所示。
  7. 一种试剂盒，包括权利要求5所述的sgRNA组合或权利要求6所述的质粒组合；所述试剂盒的用途为如下(a)或(b)或(c)：(a)制备重组细胞；(b)制备免疫缺陷动物模型；(c)制备免疫缺陷动物细胞模型。
  8. 如权利要求7所述的试剂盒，其特征在于：所述试剂盒还包括质粒pKG-GE3；
    质粒pKG-GE3中，具有特异融合基因；所述特异融合基因编码特异融合蛋白；
    所述特异融合蛋白自N端至C端依次包括如下元件：两个核定位信号、Cas9蛋白、两个核定位信号、自剪切多肽P2A、荧光报告蛋白、自裂解多肽T2A、抗性筛选标记蛋白；
    质粒pKG-GE3中，由EF1a启动子启动所述特异融合基因的表达；
    质粒pKG-GE3中，所述特异融合基因下游具有WPRE序列元件、3’LTR序列元件和bGH poly(A)signal序列元件。
  9. 权利要求5所述sgRNA组合或权利要求6所述质粒组合在制备试剂盒中的应用；所述试剂盒的用途为如下(a)或(b)或(c)：(a)制备重组细胞；(b)制备免疫缺陷动物模型；(c)制备免疫缺陷动物细胞模型。
  10. 权利要求6所述质粒组合和权利要求8中所述的质粒pKG-GE3在制备试剂盒中的应用；所述试剂盒的用途为如下(a)或(b)或(c)：(a)制备重组细胞；(b)制备免疫缺陷动物模型；(c)制备免疫缺陷动物细胞模型。
  11. 权利要求5所述sgRNA组合或权利要求6所述质粒组合或权利要求7所述试剂盒或权利要求8所述试剂盒的应用，为如下(a)或(b)或(c)：(a)制备重组细胞；(b)制备免疫缺陷动物模型；(c)制备免疫缺陷动物细胞模型。
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