WO2021159741A1 - 一种用于制备irs基因缺陷的糖尿病克隆猪核供体细胞的crispr系统及其应用 - Google Patents

一种用于制备irs基因缺陷的糖尿病克隆猪核供体细胞的crispr系统及其应用 Download PDF

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WO2021159741A1
WO2021159741A1 PCT/CN2020/124633 CN2020124633W WO2021159741A1 WO 2021159741 A1 WO2021159741 A1 WO 2021159741A1 CN 2020124633 W CN2020124633 W CN 2020124633W WO 2021159741 A1 WO2021159741 A1 WO 2021159741A1
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plasmid
irs2
sgrna
irs1
gene
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French (fr)
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牛冬
汪滔
王德华
王磊
程锐
曾为俊
马翔
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南京启真基因工程有限公司
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
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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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • 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
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • C—CHEMISTRY; METALLURGY
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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]

Definitions

  • the invention relates to a CRISPR system for preparing diabetic cloned pig nuclear donor cells with IRS gene defects and its application.
  • Diabetes diabetes mellitus, DM
  • diabetes is a metabolic disease, which is characterized by a patient's blood sugar that is higher than the standard value for a long time. Diabetes can be divided into type 1 diabetes and type 2 diabetes, among which type 2 diabetes patients account for more than 90% of the total diabetic patients.
  • Type 1 diabetes also known as insulin-dependent diabetes, is usually caused by the inability of the patient to secrete insulin due to the damage of the islet ⁇ -cells.
  • the main clinical manifestations are "three more and one less" of polyphagia, polydipsia, polyuria, and weight loss.
  • Type 2 diabetes also known as non-insulin-dependent diabetes, is usually caused by the decline of insulin's ability to regulate glucose metabolism and the decrease in insulin secretion caused by the defect of pancreatic ⁇ -cell function. Its main clinical manifestations are obesity, fatigue and weakness before the onset of disease. Without timely diagnosis, the weight will gradually decrease. Regardless of the type of diabetes, if it is not treated in time, it will cause a series of complications such as cardiovascular disease, stroke, chronic kidney disease, diabetic foot, and retinopathy, which will cause harm to the patient. According to the 2017 survey data of the International Diabetes Federation, there are currently approximately 425 million people living with diabetes worldwide, with an adult incidence rate of approximately 8.8%, of which approximately 4 million die from diabetes and its complications.
  • type 1 diabetes accounts for only about 5% of diabetic patients in my country. From a global perspective, China is even the country with the lowest prevalence of type 1 diabetes in the world, but it is a country with a high incidence of type 2 diabetes.
  • type 1 diabetes nor type 2 diabetes is controlled only by a single gene, but a complex disease that is affected by the combined effects of multiple genes and the environment. Numerous studies have shown that there are at least more than 20 different genomic regions that are closely associated with type 1 diabetes; in whole genome scanning studies of different populations, many different genomic regions that are closely associated with type 2 diabetes have also been found. In addition, numerous evidences show that the susceptibility and resistance of type 1 and type 2 diabetes have a clear tendency to familial aggregation. At present, the identified major pathogenic gene of type 1 diabetes is the human leukocyte antigen (HLA) gene located on chromosome 6, which can explain 40%-50% of the genetic susceptibility of type 1 diabetes. However, the susceptibility mechanism of type 2 diabetes is quite different in different populations. At present, it is believed that the defects of the genes encoding insulin receptor substrate protein IRS-1 and IRS-2 are closely related to type 2 diabetes.
  • HLA human leukocyte antigen
  • diabetes there is no cure for diabetes.
  • the general principle of treatment is to control blood sugar and prevent complications by changing lifestyle, controlling diet, and combining certain drugs.
  • type 1 diabetes usually patients use insulin injections to reduce blood sugar; for type 2 diabetes, patients usually take oral hypoglycemic drugs to reduce blood sugar.
  • type 2 diabetes patients usually take oral hypoglycemic drugs to reduce blood sugar.
  • gene therapy has brought new possibilities for the treatment of diabetes.
  • mouse models are the main disease animal models for studying diabetes, which can be divided into experimentally induced diabetic mouse models and spontaneous diabetic mouse models.
  • studies have been done by injecting adenoviruses expressing Pdx1 and MafA into ⁇ -cell-toxin-induced diabetic mice and autoimmune non-obese diabetic mice to re-edit ⁇ cells into functional ⁇ cells. Temporarily restored the blood glucose levels of the mice.
  • pigs are the main carnivorous supply animals for humans. They are easy to be reproduced on a large scale, and their size, physiological functions and anatomical structures are similar to humans, making them an ideal human disease model animal.
  • pig models of type 2 diabetes induced by alloxan or streptozotocin have been pig models of type 2 diabetes induced by alloxan or streptozotocin, but experimentally induced animal disease models cannot fully simulate real human diseases, and spontaneous disease animal models are difficult due to the small number of individual samples. Achieve large-scale research.
  • the purpose of the present invention is to provide a CRISPR system for preparing diabetic cloned pig nuclear donor cells deficient in IRS gene and its application.
  • the present invention provides sgRNA combinations, which are as follows (a1), (a2) or (a3):
  • the present invention provides plasmid combinations as follows (b1), (b2) or (b3):
  • the present invention also provides a kit, including the sgRNA combination.
  • the present invention also provides a kit, including the plasmid combination.
  • the kit also includes the plasmid pKG-GE3.
  • the invention also protects the application of the sgRNA combination in the preparation kit.
  • the invention also protects the application of the plasmid combination in the preparation of kits.
  • the invention also protects the application of the plasmid combination and plasmid pKG-GE3 in the preparation of the kit.
  • the ratio of the total number of moles of plasmids in the combination of plasmids to the number of moles of plasmid pKG-GE3 may be 3:1.
  • kits are as follows (c1) or (c2) or (c3): (c1) preparing recombinant cells; (c2) preparing diabetic animal models; (c3) preparing diabetic animal cell models.
  • the recombinant cell is a porcine recombinant cell.
  • the transformed recipient cell of the recombinant cell is a pig cell.
  • the porcine cells may be porcine fibroblasts.
  • the pig cells may specifically be pig primary fibroblasts.
  • the pig may specifically be Congjiang Xiang pig.
  • the recombinant cell is prepared first, and then the recombinant cell is used as a nuclear transfer donor cell to obtain a cloned animal by using somatic cell cloning technology, which is a diabetic animal model.
  • the diabetic animal model can also be used to prepare the diabetic animal cell model, that is, the corresponding cells of the diabetic animal model can be isolated and used as the diabetic animal cell model.
  • the animal model is a pig model.
  • the animal cell model is a pig cell model.
  • the animal is a pig, and specifically can be a Congjiang Xiang pig.
  • the present invention also protects the application of any one of the above sgRNA combinations or any one of the above plasmid combinations or any one of the above kits in the preparation of recombinant cells.
  • the ratio of the total moles of sgRNA plasmid to the moles of Cas plasmid can be 3:1.
  • the recombinant cell is a porcine recombinant cell.
  • the transformed recipient cell of the recombinant cell is a pig cell.
  • the porcine cells may be porcine fibroblasts.
  • the pig cells may specifically be pig primary fibroblasts.
  • the pig may specifically be Congjiang Xiang pig.
  • Any of the above-mentioned recombinant cells is a cell defective in the insulin receptor substrate gene.
  • Any of the above-mentioned recombinant cells is a cell defective in the insulin receptor substrate 1 gene.
  • Any of the above-mentioned recombinant cells is a cell defective in the insulin receptor substrate 2 gene.
  • any of the above-mentioned recombinant cells is a cell with defects in both the insulin receptor substrate 1 gene and the insulin receptor substrate 2 gene.
  • the present invention also protects the application of any one of the above sgRNA combinations or any one of the above plasmid combinations or any one of the above kits in preparing an animal model of diabetes.
  • the present invention also protects the application of any one of the above sgRNA combinations or any one of the above plasmid combinations or any one of the above kits in preparing a diabetic animal cell model.
  • the recombinant cell is prepared first, and then the recombinant cell is used as a nuclear transfer donor cell to obtain a cloned animal by using somatic cell cloning technology, which is an animal model of diabetes.
  • the recombinant cell is a porcine recombinant cell.
  • the ratio of the total number of moles of sgRNA plasmid and the number of moles of Cas plasmid can be 3:1.
  • the transformed recipient cell of the recombinant cell is a pig cell.
  • the porcine cells may be porcine fibroblasts.
  • the pig cells may specifically be pig primary fibroblasts.
  • the pig may specifically be Congjiang Xiang pig.
  • the diabetic animal model can be used to prepare the diabetic animal cell model, that is, the corresponding cells of the diabetic animal model can be isolated and used as the diabetic animal cell model.
  • the animal model is a pig model.
  • the animal cell model is a pig cell model.
  • the animal is a pig cell model.
  • the animal is a pig, and specifically can be a Congjiang Xiang pig.
  • the present invention also protects a method for preparing recombinant cells, which includes the following steps: co-transfecting porcine cells with plasmid IRS1-1, plasmid IRS1-3, plasmid IRS2-2, plasmid IRS2-3 and plasmid pKG-GE3 to obtain insulin receptor Recombinant cells with mutations in the substrate 1 gene and mutations in the insulin receptor substrate 2 gene.
  • the ratio of the total number of moles of plasmid IRS1-1, plasmid IRS1-3, plasmid IRS2-2 and plasmid IRS2-3 to the number of moles of plasmid pKG-GE3 is 3:1.
  • the porcine cells may be porcine fibroblasts.
  • the pig cells may specifically be pig primary fibroblasts.
  • the pig may specifically be Congjiang Xiang pig.
  • the recombinant cell may specifically be a recombinant cell in which the insulin receptor substrate 1 gene has a heterozygous mutation (the corresponding genotype is a heterozygous mutant) and the insulin receptor substrate 2 gene has a specific mutation; the specific mutation is homozygous Mutation (the corresponding genotype is a homozygous mutant) or biallelic mutation (the corresponding genotype is a biallelic mutant).
  • the present invention also protects a method for preparing recombinant cells, which includes the following steps: co-transfecting plasmid IRS1-1, plasmid IRS1-3 and plasmid pKG-GE3 into pig cells to obtain recombinant cells with mutations in the insulin receptor substrate 1 gene .
  • the ratio of the total number of moles of plasmid IRS1-1 and plasmid IRS1-3 to the number of moles of plasmid pKG-GE3 is 3:1.
  • the mutation is a heterozygous mutation, a homozygous mutation or a biallelic mutation.
  • the porcine cells are porcine fibroblasts.
  • the pig cells are pig primary fibroblasts.
  • the pig may specifically be Congjiang Xiang pig.
  • the present invention also protects a method for preparing recombinant cells, which includes the following steps: co-transfecting plasmid IRS2-2, plasmid IRS2-3 and plasmid pKG-GE3 into pig cells to obtain recombinant cells with mutations in the insulin receptor substrate 2 gene .
  • the ratio of the total number of moles of plasmid IRS2-2 and plasmid IRS2-3 to the number of moles of plasmid pKG-GE3 is 3:1.
  • the mutation is a heterozygous mutation, a homozygous mutation or a biallelic mutation.
  • the porcine cells are porcine fibroblasts.
  • the pig cells are pig primary fibroblasts.
  • the pig may specifically be Congjiang Xiang pig.
  • the present invention also protects the recombinant cells prepared by any of the above methods.
  • the recombinant cell may be a recombinant cell described in any one of Table 3, Table 4, or Table 5.
  • the recombinant cell may be any one of the following: a monoclonal cell numbered 3, 10, 16, 22, 4, 5, 6, 7, 17 or 24 in Table 3.
  • the recombinant cell may be any one of the following: a monoclonal cell numbered 25, 28, 30, 35, 47, 33, 37, 41, or 48 in Table 4.
  • the recombinant cell may be any one of the following: a monoclonal cell numbered 51, 66, or 70 in Table 5.
  • the present invention also protects the application of the recombinant cell in preparing an animal model of diabetes.
  • the invention also protects the application of the recombinant cell in preparing a diabetic animal cell model.
  • the recombinant cell is prepared first, and then the recombinant cell is used as a nuclear transfer donor cell to obtain a cloned animal by using somatic cell cloning technology, which is a diabetic animal model.
  • the diabetic animal model can be used to prepare the diabetic animal cell model, that is, the corresponding cells of the diabetic animal model can be isolated and used as the diabetic animal cell model.
  • the animal model is a pig model.
  • the animal cell model is a pig cell model.
  • the animal is a pig, and specifically can be a Congjiang Xiang pig.
  • Any of the above-mentioned diabetes may specifically be type 2 diabetes.
  • the target sequence binding region of the sgRNA IRS1-1 is shown in nucleotides 1-20 in SEQ ID NO:8.
  • the sgRNA IRS1-1 is specifically shown in SEQ ID NO:8.
  • the target sequence binding region of the sgRNA IRS1-3 is shown in nucleotides 1-20 in SEQ ID NO:10.
  • the sgRNA IRS1-3 is specifically shown in SEQ ID NO:10.
  • the target sequence binding region of the sgRNA IRS2-2 is shown in nucleotides 1-20 in SEQ ID NO:14.
  • the sgRNA IRS2-2 is specifically shown in SEQ ID NO:14.
  • the target sequence binding region of the sgRNA IRS2-3 is shown in nucleotides 1-20 in SEQ ID NO:15.
  • the sgRNA IRS2-3 is specifically shown in SEQ ID NO:15.
  • the plasmid IRS1-1 was transcribed to obtain sgRNA IRS1-1 .
  • the plasmid IRS1-3 was transcribed to obtain sgRNA IRS1-3 .
  • the plasmid IRS2-2 is transcribed to obtain sgRNA IRS2-2 .
  • the plasmid IRS2-3 was transcribed to obtain sgRNA IRS2-3 .
  • the plasmid is by means of IRS1-1 BbsI restriction endonuclease target sequence encoding the binding domain sgRNA IRS1-1 insertion pKG-U6gRNA vector obtained.
  • the plasmid is IRS1-3 by restriction enzyme BbsI within the coding sequence of the target sequence-binding region sgRNA IRS1-3 insertion pKG-U6gRNA vector obtained.
  • the plasmid is the plasmid IRS2-2 by restriction enzyme BbsI endo target sequence encoding the binding domain sgRNA IRS2-2 insertion pKG-U6gRNA vector obtained.
  • the plasmid is IRS2-3 by restriction enzyme BbsI within the coding sequence of the target sequence-binding region sgRNA IRS2-3 insertion pKG-U6gRNA vector obtained.
  • the plasmid pKG-GE3 has a specific fusion gene; the specific fusion gene encodes a specific fusion protein;
  • the specific fusion protein includes the following elements in sequence 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- Sex screening marker protein;
  • the EF1a promoter initiates the expression of the specific fusion gene
  • the downstream of the specific fusion gene 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, bGH poly(A) signal sequence element.
  • the two nuclear localization signals upstream of the Cas9 protein are SV40 nuclear localization signals
  • the two nuclear localization signals downstream of the 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 Puromycin protein.
  • the amino acid sequence of the self-cleaving polypeptide P2A is "ATNFSLLKQAGDVEENPGP" (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 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 in 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 element is shown in nucleotides 6722-7310 in SEQ ID NO: 2.
  • the 3'LTR sequence element is shown in nucleotides 7382-7615 in SEQ ID NO: 2.
  • the bGH poly(A) signal sequence element is shown in nucleotides 7647-7871 in SEQ ID NO: 2.
  • the plasmid pKG-GE3 is specifically shown in SEQ ID NO: 2.
  • the plasmid pKG-U6gRNA is specifically shown in SEQ ID NO: 3.
  • Pig IRS1 gene information is 100512686, Sus scrofa.
  • the pig IRS1 gene encodes insulin receptor substrate 1.
  • the protein encoded by the pig IRS1 gene is shown in SEQ ID NO: 4.
  • the pig IRS1 gene has 2 exons, the first exon is shown in SEQ ID NO: 6, and the second exon is shown in SEQ ID NO: 7.
  • the open reading frame of the porcine IRS1 gene is shown in nucleotides 1-3726 in SEQ ID NO: 6.
  • the IRS1 gene is a gene encoding the protein shown in SEQ ID NO:4.
  • the IRS1 gene is a gene with a DNA molecule shown in SEQ ID NO:6.
  • the IRS1 gene is the pig IRS1 gene.
  • Pig IRS2 gene information is 110255858, Sus scrofa.
  • the pig IRS2 gene encodes insulin receptor substrate 2.
  • the protein encoded by the porcine IRS2 gene is shown in SEQ ID NO: 11.
  • genomic DNA the porcine IRS2 gene has 2 exons, the coding region of the first exon is shown in SEQ ID NO: 12 nucleotides 1-4006, and the coding region of the second exon This is shown in nucleotides 4007-4011 in SEQ ID NO: 12.
  • the open reading frame of the pig IRS2 gene is shown in SEQ ID NO: 12.
  • the IRS2 gene is a gene encoding the protein shown in SEQ ID NO: 11.
  • the IRS2 gene is a gene with a DNA molecule shown in SEQ ID NO: 12.
  • the IRS2 gene is a pig IRS2 gene.
  • the present invention has at least the following beneficial effects:
  • the present invention uses a double gRNA combination for mutation. Compared with single gRNA, it can effectively reduce the generation of non-frameshift mutations, and PCR can be directly used to detect gene editing efficiency. If a single gRNA is used to mutate the target gene, in the random repair of DNA non-homologous end joining (NHEJ), there will be a 1/3 probability of generating base non-frameshift mutations, and non-frameshift mutations may not be destroyed. The function of the target gene fails to achieve the expected goal of inactivating the target gene.
  • NHEJ DNA non-homologous end joining
  • a fragment of the target gene can be removed, and by designing to remove non-three-fold base fragments, the fragment deletion frameshift mutation of the target gene can be effectively generated.
  • the gene editing product of the missing fragment can also be detected directly by PCR, and the efficiency of gene editing can be directly estimated by the ratio of the gene editing product to the wild-type product (ie, the unedited product).
  • the efficiency of gene editing can be directly estimated by the ratio of the gene editing product to the wild-type product (ie, the unedited product).
  • the gRNA vector and the cas9 vector are not based on the conventional 1:1 molar ratio, but based on the 3:1 molar ratio.
  • the most suitable amount of the two gRNA plasmids and the Cas9 plasmid is a molar ratio of 1.5:1.5:1, and the actual amount of plasmids is 0.46ug+0.46ug+1.08ug.
  • the time for gRNA vector to transcribe gRNA is earlier than the time for cas9 protein formation, and the transcribed gRNA degrades quickly.
  • the present invention preferably adopts a carrier molar ratio of 3:1 gRNA:cas9.
  • the research object (pig) of the present invention has better applicability than other animals (rats, mice, primates). So far, only spontaneous mutant mouse diabetic disease models have been selected, and no large animal diabetic disease models have been successfully developed. Rodents such as rats and mice are very different from humans in terms of body size, organ size, physiology, pathology, etc., and cannot truly simulate the normal physiological and pathological conditions of humans. Studies have shown that more than 95% of drugs that have been validated in rats and mice are ineffective in human clinical trials. As far as large animals are concerned, primates are the animals closest to humans, but they are small in size, late in sexual maturity (mating at the age of 6-7), and are singleton animals, their population expansion speed is extremely slow, and the cost of breeding Also high.
  • pigs do not have the above-mentioned shortcomings. Pigs are the closest relatives to humans except primates. Their body size, weight, organ size, etc. are similar to humans, and they are in anatomy, physiology, nutritional metabolism, disease pathogenesis, etc. It is very similar to human beings. At the same time, pigs have early sexual maturity (4-6 months), high fecundity, multiple litters, and a larger group can be formed within 2-3 years.
  • pig cloning technology is very mature, and the cost of cloning and feeding is much lower than that of primates; moreover, pigs have long been a carnivorous animal for humans, and the use of pigs as disease model animals has relatively little resistance in terms of animal protection and ethics. .
  • the cas9 high-efficiency expression vector modified by the present invention is used for gene editing, and the editing efficiency is 3 to 4 times higher than that of the original vector.
  • 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-U6gRNA.
  • Fig. 4 is a schematic diagram of inserting a DNA molecule of about 20 bp (for transcription to form the target sequence binding region of gRNA) into the plasmid pKG-U6gRNA.
  • Fig. 5 is an electrophoresis diagram after PCR amplification using the genomic DNA of 8 pigs as a template and using a primer pair composed of primers IRS1-GT-F412/IRS1-GT-R1220 in step 1 of Example 2.
  • Fig. 6 shows various double-stranded DNA molecules with sticky ends in step 3 of Example 2.
  • Fig. 7 is an electrophoresis diagram after PCR amplification of genomic DNA using a primer pair composed of IRS1-F583 and IRS1-R961 in step 4 of Example 2.
  • Fig. 8 is an electrophoresis diagram of PCR amplification using the genomic DNA of 8 pigs as a template and using a primer pair consisting of primers IRS2-GT-nF848/IRS2-GT-nR1710.
  • Fig. 10 is an electrophoresis diagram after PCR amplification of genomic DNA using a primer pair composed of IRS2-GT-nF848 and IRS2-GT-nR1502 in step 4 of Example 3.
  • FIG. 11 is an electrophoresis diagram of the cells obtained in the first group in Example 4.
  • FIG. 12 is an electrophoresis diagram of the cells obtained in the second group in Example 4.
  • Figure 13 is an electrophoresis image of the cells obtained in the third group in Example 4 (primer pair composed of IRS1-F583 and IRS1-R961).
  • Fig. 14 is an electrophoresis diagram of the cells obtained in the third group in Example 4 (primer pair composed of IRS2-GT-nF848 and IRS2-GT-nR1502).
  • FIG. 15 is the sequencing peak diagram of IRS1-3.
  • FIG. 16 shows the sequencing peak diagram of IRS1-4.
  • Figure 17 is a sequencing peak diagram of IRS2-25.
  • Figure 18 is a sequencing peak diagram of IRS2-26.
  • FIG. 19 is an electrophoresis diagram after PCR amplification with a primer pair composed of MSTN-F896 and MSTN-R1351 using genomic DNA as a template in step 2 of Example 5.
  • FIG. 20 is an electrophoresis diagram of three groups of MSTN in step three of Example 5.
  • FIG. 20 is an electrophoresis diagram of three groups of MSTN in step three of Example 5.
  • 21 is an electrophoresis diagram of the three groups of FNDC5 in step 3 of Example 5.
  • the 8 pigs in the examples are all newly born Congjiangxiang pigs, including 4 females (named 1, 2, 3, 4 respectively) and 4 males (named A, B, C, and D respectively).
  • the method of preparing pig primary fibroblasts 1 Take 0.5g of pig ear tissue, remove hair, then soak in 75% alcohol for 30-40s, and then wash with PBS buffer containing 5% (volume ratio) Penicillin-Streptomycin (Gibco) 5 times, and then washed once with PBS buffer; 2Cut the tissue into small pieces with scissors, digest with 5mL 1% collagenase solution (Sigma), digest for 1h at 37°C, then centrifuge at 500g for 5min, discard the supernatant; 3Use 1mL of the pellet to complete Resuspend the culture medium, and then spread it in a 9cm diameter cell culture dish containing 10mL of complete medium and sealed with 0.2% gelatin (VWR), and cultivate until the cells are about 60% full of the bottom of the dish; 4After step 3 is completed , Use trypsin to digest and collect cells, use cell cryopreservation solution (90% complete medium + 10% DMSO, volume ratio) to freeze the cells.
  • PBS buffer
  • the pig primary fibroblasts used in Examples 2 to 5 were all obtained from the above-mentioned pig named 2 (female, blood type AO).
  • Plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 referred to as plasmid pX330.
  • Plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO referred to as plasmid pKG-GE3.
  • Plasmid pX330, plasmid pKG-GE3, plasmid pKG-U6gRNA are all circular plasmids.
  • nucleotides 440-725 constitute the CMV enhancer
  • nucleotides 727-1208 constitute the chicken ⁇ -actin promoter
  • nucleotides 1304-1324 encode the SV40 nuclear localization signal (NLS )
  • nucleotides 1325-5449 encode Cas9 protein
  • nucleotides 5450-5497 encode nucleoplasmin nuclear localization signal (NLS).
  • nucleotides 395-680 constitute the CMV enhancer
  • nucleotides 682-890 constitute the EF1a promoter
  • nucleotides 986-1006 encode the nuclear localization signal (NLS)
  • nucleotides 682-890 constitute the 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)
  • nucleotides 5219-5266 The acid encodes the nuclear localization signal (NLS)
  • nucleotides 5276-5332 encode the self-splicing polypeptide P2A (the amino acid sequence of the self-splicing polypeptide P2A is "ATNFSLLKQAGDVEENPGP", and the cleavage position that occurs from the splicing is the first from the C-terminus Between two amino acid residues and the second amino acid residue), the 5333-6046th nucleotides encode the EGFP protein, and the 6056-6109th nucleotides encode the self-cleaving polypeptide T2A (the amino acid sequence of the self-cleaving polypeptide T2A is " EGRGSLLTCGDVEENPGP", the position of
  • Nos. 911-6706 form a fusion gene and express the fusion protein. Due to the existence of the self-cleaving polypeptide P2A and the self-cleaving polypeptide T2A, the fusion protein spontaneously forms the following three proteins: the protein with the Cas9 protein, the protein with the EGFP protein, and the protein with the Puro protein.
  • the plasmid pKG-GE3 is mainly modified as follows: 1Removal of 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 coding gene (NLS) in the upstream and downstream of Cas9 gene to increase the nuclear localization ability of Cas9 protein; 4The original plasmid does not have any eukaryotic selection markers, which is not conducive to positive For the selection and enrichment of transformed cells, insert the P2A-EGFP-T2A-PURO coding gene in the downstream of Cas9 gene to give the vector fluorescence and eukaryotic resistance screening ability; 5Insert WPRE element and 3'LTR sequence element to enhance Cas9 The protein translation ability of genes
  • Figure 3 shows a schematic diagram of the structure of plasmid pKG-U6gRNA.
  • nucleotides 2280-2539 constitute the hU6 promoter, and nucleotides 2558-2637 are used for transcription to form a gRNA backbone.
  • a DNA molecule of about 20 bp (used to transcribe the target sequence binding region of gRNA) is inserted into the plasmid pKG-U6gRNA to form a recombinant plasmid.
  • the schematic diagram is shown in Figure 4, and the recombinant plasmid is transcribed to obtain gRNA in the cell.
  • Pig IRS1 gene information encoding insulin receptor substrate 1; located on chromosome 15; GeneID is 100512686, Sus scrofa.
  • the protein encoded by the pig IRS1 gene is shown in SEQ ID NO: 4.
  • the pig IRS1 gene has 2 exons, the first exon is shown in SEQ ID NO: 6, and the second exon is shown in SEQ ID NO: 7.
  • the genomic DNA the partial nucleotide located upstream of the first exon of the pig IRS1 gene is shown in SEQ ID NO: 5.
  • the open reading frame of the porcine IRS1 gene is located in the first exon, as shown by nucleotides 1-3726 in SEQ ID NO: 6.
  • PCR amplification was performed using a primer pair composed of primers IRS1-GT-F412/IRS1-GT-R1220, and then electrophoresis was performed, as shown in Figure 5.
  • the PCR amplification products are recovered and sequenced, and the sequencing results are compared and analyzed with the IRS1 gene sequence in the public database. According to the comparison results, design primers for detecting mutations (the primers themselves avoid possible mutation sites).
  • the primers designed to detect mutations are: IRS1-F583 and IRS1-R961.
  • IRS1-GT-F412 5’-GCATGAAACGCCAGTAAACTCCG-3’;
  • IRS1-GT-R1220 5'-CGAAACTGATGGTCTTGCTGGTC-3'.
  • IRS1-F583 5’-CCACCCGGTTGTTTTTCGGCG-3’;
  • IRS1-R961 5’-CTGGTACCAGCTGTCCTGTTCG-3’.
  • the three targets are as follows:
  • sgRNA IRS1-1 target 5'-CTCGTAGTACTCGAGGCGCG-3';
  • sgRNA IRS1-2 target 5'-ATGTTGAAGCAGCTCTCCAG-3';
  • sgRNA IRS1-3 target 5'-GCTGCTTCAACATCAACAAG-3'.
  • IRS1-1S and IRS1-1A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends (Figure 6A). Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid pKG-U6gRNA (IRS1-1). The plasmid pKG-U6gRNA (IRS1-1) expresses the sgRNA IRS1-1 shown in SEQ ID NO:8.
  • IRS1-2S and IRS1-2A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends (Figure 6B). Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid pKG-U6gRNA (IRS1-2).
  • the plasmid pKG-U6gRNA (IRS1-2) expresses the sgRNA IRS1-2 shown in SEQ ID NO:9.
  • IRS1-3S and IRS1-3A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends (Figure 6C). Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid pKG-U6gRNA (IRS1-3).
  • the plasmid pKG-U6gRNA (IRS1-3) expresses the sgRNA IRS1-3 shown in SEQ ID NO:10.
  • IRS1-1S 5’-caccgCTCGTAGTACTCGAGGCGCG-3’;
  • IRS1-1A 5’-aaacCGCGCCTCGAGTACTACGAGc-3’.
  • IRS1-2S 5’-caccgATGTTGAAGCAGCTCTCCAG-3’;
  • IRS1-2A 5’-aaacCTGGAGAGCTGCTTCAACATc-3’.
  • IRS1-3S 5’-caccGCTGCTTCAACATCAACAAG-3’;
  • IRS1-3A 5'-aaacCTTGTTGATGTTGAAGCAGC-3'.
  • IRS1-1S, IRS1-1A, IRS1-2S, IRS1-2A, IRS1-3S, IRS1-3A are all single-stranded DNA molecules.
  • Plasmid pKG-U6gRNA (IRS1-1), plasmid pKG-U6gRNA (IRS1-2) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts.
  • Mixing ratio about 200,000 pig primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (IRS1-1): 0.46 ⁇ g plasmid pKG-U6gRNA (IRS1-2): 1.08 ⁇ g plasmid pKG-GE3.
  • Plasmid pKG-U6gRNA (IRS1-1), plasmid pKG-U6gRNA (IRS1-3) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts.
  • Mixing ratio about 200,000 pig primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (IRS1-1): 0.46 ⁇ g plasmid pKG-U6gRNA (IRS1-3): 1.08 ⁇ g plasmid pKG-GE3.
  • the third group pig primary fibroblasts, without any transfection operation.
  • Co-transfection adopts electroporation transfection method, using mammalian nuclear transfection kit (Neon kit, Thermofisher) and NeonTM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use complete culture medium to incubate for 16-18 hours, and then replace with new complete medium for culture.
  • the total culture time is 48 hours.
  • step 2 digest and collect cells with trypsin, extract genomic DNA, use primer pair composed of IRS1-F583 and IRS1-R961 for PCR amplification, and then perform electrophoresis.
  • the results are shown in Figure 7.
  • the larger band is the wild-type band (WT), and the smaller band is the mutant band (MT).
  • Gene deletion mutation efficiency (MT grayscale/MT band bp number)/(WT grayscale/WT band bp number+MT grayscale/MT band bp number) ⁇ 100%.
  • the first group of gene deletion mutation efficiency is 57%
  • the second group of gene deletion mutation efficiency is 65%
  • the third group of gene deletion mutation efficiency is 0%.
  • Pig IRS2 gene information encoding insulin receptor substrate 2; located on chromosome 11; GeneID is 110255858, Sus scrofa.
  • the protein encoded by the porcine IRS2 gene is shown in SEQ ID NO: 11.
  • genomic DNA the porcine IRS2 gene has 2 exons, the coding region of the first exon is shown in SEQ ID NO: 12 nucleotides 1-4006, and the coding region of the second exon This is shown in nucleotides 4007-4011 in SEQ ID NO: 12.
  • PCR amplification was performed using a primer pair composed of primers IRS2-GT-nF848/IRS2-GT-nR1710, and then electrophoresis was performed, as shown in Figure 8.
  • the PCR amplification products are recovered and sequenced, and the sequencing results are compared and analyzed with the IRS2 gene sequence in the public database. According to the comparison results, design primers for detecting mutations (the primers themselves avoid possible mutation sites).
  • the primers designed to detect mutations are: IRS2-GT-nF848 and IRS2-GT-nR1502.
  • IRS2-GT-nF848 5’-AGAACATCCACGAGACCATCCTG-3’;
  • IRS2-GT-nR1710 5’-TCTCAGCCCTCTATCCAAGTCCT-3’;
  • IRS2-GT-nR1502 5'-TCATCCAGGGACATAAAGCCAGG-3'.
  • the four targets are as follows:
  • sgRNA IRS2-1 target 5'-GACGACTGGCTCTTGCTGCG-3';
  • sgRNA IRS2-2 target 5'-GGTTGACCAGGTGGTGGTGG-3';
  • sgRNA IRS2-3 target 5'-CACGAGCTGCACTTGGCCGC-3';
  • IRS2-1S and IRS2-1A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends (Figure 9A). Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid pKG-U6gRNA (IRS2-1).
  • the plasmid pKG-U6gRNA (IRS2-1) expresses the sgRNA IRS2-1 shown in SEQ ID NO:13.
  • IRS2-2S and IRS2-2A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends (Figure 9B). Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid pKG-U6gRNA (IRS2-2).
  • the plasmid pKG-U6gRNA (IRS2-2) expresses the sgRNA IRS2-2 shown in SEQ ID NO:14.
  • IRS2-3S and IRS2-3A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends (Figure 9C). Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid pKG-U6gRNA (IRS2-3).
  • the plasmid pKG-U6gRNA (IRS2-3) expresses the sgRNA IRS2-3 shown in SEQ ID NO: 15.
  • IRS2-4S and IRS2-4A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends (Figure 9D). Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid pKG-U6gRNA (IRS2-4).
  • the plasmid pKG-U6gRNA (IRS2-4) expresses the sgRNA IRS2-4 shown in SEQ ID NO:16.
  • IRS2-1S 5’-caccGACGACTGGCTCTTGCTGCG-3’;
  • IRS2-1A 5'-aaacCGCAGCAAGAGCCAGTCGTC-3'.
  • IRS2-2S 5’-caccGGTTGACCAGGTGGTGGTGG-3’;
  • IRS2-2A 5’-aaacCCACCACCACCACCTGGTCAACC-3’.
  • IRS2-3S 5’-caccgCACGAGCTGCACTTGGCCGC-3’;
  • IRS2-3A 5'-aaacGCGGCCAAGTGCAGCTCGTGc-3'.
  • IRS2-4S 5’-caccgAACCCGGCACGAGCTGCACT-3’;
  • IRS2-4A 5'-aaacAGTGCAGCTCGTGCCGGGTTc-3'.
  • IRS2-1S, IRS2-1A, IRS2-2S, IRS2-2A, IRS2-3S, IRS2-3A, IRS2-4S, IRS2-4A are all single-stranded DNA molecules.
  • Plasmid pKG-U6gRNA (IRS2-1), plasmid pKG-U6gRNA (IRS2-2) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts.
  • Plasmid pKG-U6gRNA (IRS2-1), plasmid pKG-U6gRNA (IRS2-4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts.
  • Plasmid pKG-U6gRNA (IRS2-2), plasmid pKG-U6gRNA (IRS2-3) and plasmid pKG-GE3 were co-transfected into pig primary fibroblasts.
  • Plasmid pKG-U6gRNA (IRS2-2), plasmid pKG-U6gRNA (IRS2-4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts.
  • the fifth group pig primary fibroblasts, without any transfection operation.
  • Co-transfection adopts electroporation transfection method, using mammalian nuclear transfection kit (Neon kit, Thermofisher) and NeonTM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use complete culture medium to incubate for 16-18 hours, and then replace with new complete medium for culture.
  • the total culture time is 48 hours.
  • step 2 digest and collect cells with trypsin, extract genomic DNA, use primer pair composed of IRS2-GT-nF848 and IRS2-GT-nR1502 for PCR amplification, and then perform electrophoresis.
  • the results are shown in Figure 10.
  • the larger band is the wild-type band (WT), and the smaller band is the mutant band (MT).
  • Gene deletion mutation efficiency (MT grayscale/MT band bp number)/(WT grayscale/WT band bp number+MT grayscale/MT band bp number) ⁇ 100%.
  • the first group of gene deletion mutation efficiency is 50%
  • the second group of gene deletion mutation efficiency is undetected
  • the third group of gene deletion mutation efficiency is 91%.
  • the efficiency of gene deletion and mutation in the fourth group was 82%
  • the efficiency of gene deletion and mutation in the fifth group (negative control group) was 0%.
  • Plasmid pKG-U6gRNA (IRS1-1), plasmid pKG-U6gRNA (IRS1-3) and plasmid pKG-GE3 were co-transfected into pig primary fibroblasts.
  • Mixing ratio about 200,000 pig primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (IRS1-1): 0.46 ⁇ g plasmid pKG-U6gRNA (IRS1-3): 1.08 ⁇ g plasmid pKG-GE3.
  • Plasmid pKG-U6gRNA (IRS2-2), plasmid pKG-U6gRNA (IRS2-3) and plasmid pKG-GE3 were co-transfected into pig primary fibroblasts.
  • the third group Plasmid pKG-U6gRNA (IRS1-1), plasmid pKG-U6gRNA (IRS1-3), plasmid pKG-U6gRNA (IRS2-2), plasmid pKG-U6gRNA (IRS2-3) and plasmid pKG-GE3 Transfection of pig primary fibroblasts.
  • Proportion about 200,000 pig primary fibroblasts: 0.23 ⁇ g plasmid pKG-U6gRNA(IRS1-1): 0.23 ⁇ g plasmid pKG-U6gRNA(IRS1-3): 0.23 ⁇ g plasmid pKG-U6gRNA(IRS2-2): 0.23 ⁇ g plasmid pKG-U6gRNA (IRS2-3): 1.08 ⁇ g plasmid pKG-GE3.
  • Co-transfection adopts electroporation transfection method, using mammalian nuclear transfection kit (Neon kit, Thermofisher) and NeonTM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use complete culture medium to incubate for 16-18 hours, and then replace with new complete medium for culture.
  • the total culture time is 48 hours.
  • step 3 use trypsin to digest and collect the cells, then wash with complete culture medium, then resuspend in complete medium, and then pick each single clone and transfer to a 96-well plate (1 cell per well) , Each well is equipped with 200 ⁇ l complete culture medium), culture for 2 weeks (replace with new complete medium every 2-3 days).
  • step 3 digest and collect cells with trypsin (about 2/3 of the cells obtained in each well are inoculated into a 6-well plate containing complete culture medium, and the remaining 1/3 are 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 cryopreservation solution (90% complete medium + 10% DMSO, volume ratio) live.
  • step 6 Take the centrifuge tube of step 4, take the cells, extract genomic DNA, and perform PCR amplification (the cells obtained in the first group are amplified by the primer pair composed of IRS1-F583 and IRS1-R961, and the cells obtained in the second group are amplified by PCR.
  • the primer pair composed of IRS2-GT-nF848 and IRS2-GT-nR1502 was used for PCR amplification, and the cells obtained in the third group were respectively used for PCR amplification using the above two primer pairs, and then subjected to electrophoresis. Porcine primary fibroblasts were used as wild-type control.
  • the electropherogram of the cells obtained in the first group is shown in Figure 11.
  • the electropherogram of the cells obtained in the second group is shown in Figure 12.
  • Figure 13 shows the electropherogram of the cells obtained in the third group (primer pair composed of IRS1-F583 and IRS1-R961).
  • the electropherogram of the cells obtained in the third group is shown in Figure 14.
  • the lane numbers are consistent with the monoclonal cell numbers.
  • step 6 the PCR amplified 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 sequencing results of a certain monoclonal cell, one is consistent with the sequencing results of pig primary fibroblasts, and the other has mutations compared with the sequencing results of pig primary fibroblasts (the mutations include one or more Nucleotide deletion, insertion or substitution), the genotype of the monoclonal cell is heterozygous; if the sequencing results of a certain monoclonal cell are two, both are compared with the sequencing results of porcine primary fibroblasts.
  • Mutation includes deletion, insertion or substitution of one or more nucleotides
  • the genotype of the monoclonal cell is a biallelic mutant
  • mutations have occurred (mutations include deletion, insertion or substitution of one or more nucleotides)
  • the genotype of the monoclonal cell is a homozygous mutant; if a certain monoclonal cell is sequenced The result is one, and consistent with the sequencing result of pig primary fibroblasts, the genotype of this monoclonal cell is homozygous wild type.
  • the results of the first group are shown in Table 3.
  • the genotypes of monoclonal cells numbered 3, 10, 16, 22 are homozygous mutants.
  • the genotypes of monoclonal cells numbered 4, 5, 6, 7, 17, and 24 are biallelic mutants.
  • the genotypes of monoclonal cells numbered 8, 11, 13, 15, 19, 20, and 23 are heterozygous.
  • the genotypes of the monoclonal cells numbered 1, 2, 9, 12, 14, 18, 21 are homozygous wild-type.
  • the ratio of IRS1 gene-edited monoclonal cells was 17/24.
  • the sequencing peak diagram of IRS1-3 is shown in Figure 15, and the sequencing peak diagram of IRS1-4 is shown in Figure 16.
  • the results of the second group are shown in Table 4.
  • the genotypes of monoclonal cells numbered 25, 28, 30, 35, and 47 are homozygous mutants.
  • the genotypes of monoclonal cells numbered 33, 37, 41, and 48 are biallelic mutants.
  • the genotypes of monoclonal cells numbered 26, 34, 36, 38, 39, 42, 43, 45 are heterozygous.
  • the genotypes of monoclonal cells numbered 27, 29, 31, 32, 40, 44, and 46 are homozygous wild-type.
  • the ratio of IRS2 gene-edited monoclonal cells was 17/24.
  • the sequencing peak diagram of IRS2-25 is shown in Figure 17, and the sequencing peak diagram of IRS2-26 is shown in Figure 18.
  • the target of MSTN-gRNA2 5'-TTTCCAGGCGAAGTTTACTG-3'.
  • FNDC5-gRNA1 5’-TGTACTCAGTGTCCTCCTCC-3’;
  • FNDC5-gRNA2 5'-GCTCTTCAAGACGCCTCGCG-3'.
  • MSTN-F896 5’-TCTCTCAGACAGTGCAGGCATTA-3’;
  • MSTN-R1351 5'-CGTTTCCGTCGTAGCGTGATAAT-3'.
  • FNDC5-F209 5’-CAGTTCTCACTTGATGGCCTTGG-3’;
  • FNDC5-R718 5'-AGGGGTCTGGGGAGGAATGG-3'.
  • MSTN-1S and MSTN-1A respectively, then mix and anneal to obtain double-stranded DNA molecules with sticky ends. Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid pKG-U6gRNA (MSTN-1).
  • MSTN-2S and MSTN-2A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid pKG-U6gRNA (MSTN-2).
  • FNDC5-1S and FNDC5-1A respectively, then mix and anneal to obtain double-stranded DNA molecules with sticky ends. Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid pKG-U6gRNA (FNDC5-1).
  • FNDC5-2S and FNDC5-2A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. Connect the double-stranded DNA molecule with sticky ends to the vector backbone to obtain the plasmid 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'.
  • the first group Plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts.
  • Mixing ratio about 200,000 pig primary fibroblasts: 0.22 ⁇ g plasmid pKG-U6gRNA (MSTN-1): 0.22 ⁇ g plasmid pKG-U6gRNA (MSTN-2): 1.56 ⁇ g plasmid pKG-GE3.
  • the molar ratio of plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pKG-GE3 is 0.5:0.5:1 in sequence.
  • the second group Plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pKG-GE3 were co-transfected into pig primary fibroblasts.
  • Mixing ratio about 200,000 pig primary fibroblasts: 0.36 ⁇ g plasmid pKG-U6gRNA (MSTN-1): 0.36 ⁇ g plasmid pKG-U6gRNA (MSTN-2): 1.27 ⁇ g plasmid pKG-GE3. That is, the molar ratio of plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pKG-GE3 is 1:1:1 in sequence.
  • the third group Plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pKG-GE3 were co-transfected into pig primary fibroblasts. Proportion: about 200,000 pig primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (MSTN-1): 0.46 ⁇ g plasmid pKG-U6gRNA (MSTN-2): 1.08 ⁇ g plasmid pKG-GE3. That is, the molar ratio of plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pKG-GE3 is 1.5:1.
  • the fourth group Plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pKG-GE3 were co-transfected into pig primary fibroblasts.
  • Mixing ratio about 200,000 pig primary fibroblasts: 0.53 ⁇ g plasmid pKG-U6gRNA (MSTN-1): 0.53 ⁇ g plasmid pKG-U6gRNA (MSTN-2): 0.93 ⁇ g plasmid pKG-GE3. That is, the molar ratio of plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pKG-GE3 is 2:2:1.
  • the fifth group Plasmid pKG-U6gRNA (MSTN-1) and plasmid pKG-U6gRNA (MSTN-2) were co-transfected into porcine primary fibroblasts. Mixing ratio: about 200,000 pig primary fibroblasts: 1 ⁇ g plasmid pKG-U6gRNA (MSTN-1): 1 ⁇ g plasmid pKG-U6gRNA (MSTN-2).
  • Co-transfection adopts electroporation transfection method, using mammalian nuclear transfection kit (Neon kit, Thermofisher) and NeonTM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use complete culture medium to incubate for 16-18 hours, and then replace with new complete medium for culture.
  • the total culture time is 48 hours.
  • step 2 digest and collect the cells with trypsin, extract genomic DNA, use a primer pair consisting of MSTN-F896 and MSTN-R1351 for PCR amplification, and then perform electrophoresis.
  • the electrophoresis result is shown in Figure 19.
  • the 456bp band is a wild-type band (WT), and about 329bp (theoretical deletion of band 456bp is 127bp) is a deletion mutation band (MT).
  • WT wild-type band
  • MT deletion mutation band
  • Gene deletion mutation efficiency (MT grayscale/MT band bp number)/(WT grayscale/WT band bp number+MT grayscale/MT band bp number) ⁇ 100%.
  • the efficiency of gene deletion mutation in the first group was 28.6%
  • the efficiency of gene deletion and mutation in the second group was 77.8%
  • the efficiency of gene deletion in the third group was 86.8%
  • the efficiency of gene deletion and mutation in the fourth group was 81.5%.
  • the third group has the highest gene editing efficiency. It is determined that the most suitable amount of the two gRNA plasmids and the Cas9 plasmid is 1.5:1.
  • MSTN-B group The plasmid pKG-U6gRNA (MSTN-1) and plasmid pKG-U6gRNA (MSTN-2) were co-transfected into pig primary fibroblasts. Mixing ratio: about 200,000 pig primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (MSTN-1): 0.46 ⁇ g plasmid pKG-U6gRNA (MSTN-2).
  • MSTN-330 group The plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pX330 were co-transfected into porcine primary fibroblasts. Mixing ratio: about 200,000 pig primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (MSTN-1): 0.46 ⁇ g plasmid pKG-U6gRNA (MSTN-2): 1.08 ⁇ g plasmid pX330.
  • MSTN-KG group The plasmid pKG-U6gRNA (MSTN-1), plasmid pKG-U6gRNA (MSTN-2) and plasmid pKG-GE3 were co-transfected into pig primary fibroblasts. Mixing ratio: about 200,000 pig 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 The plasmid pKG-U6gRNA (FNDC5-1) and plasmid pKG-U6gRNA (FNDC5-2) were co-transfected into pig primary fibroblasts. Mixing ratio: about 200,000 pig primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (FNDC5-1): 0.46 ⁇ g plasmid pKG-U6gRNA (FNDC5-2).
  • FNDC5-330 group The plasmid pKG-U6gRNA (FNDC5-1), plasmid pKG-U6gRNA (FNDC5-2) and plasmid pX330 were co-transfected into pig primary fibroblasts. Mixing ratio: about 200,000 pig primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (FNDC5-1): 0.46 ⁇ g plasmid pKG-U6gRNA (FNDC5-2): 1.08 ⁇ g plasmid pX330.
  • FNDC5-KG group The plasmid pKG-U6gRNA (FNDC5-1), plasmid pKG-U6gRNA (FNDC5-2) and plasmid pKG-GE3 were co-transfected into pig primary fibroblasts. Mixing ratio: about 200,000 pig primary fibroblasts: 0.46 ⁇ g plasmid pKG-U6gRNA (FNDC5-1): 0.46 ⁇ g plasmid pKG-U6gRNA (FNDC5-2): 1.08 ⁇ g plasmid pKG-GE3.
  • Co-transfection adopts electroporation transfection method, using mammalian nuclear transfection kit (Neon kit, Thermofisher) and NeonTM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).
  • step 2 After completing step 1, use complete culture medium to incubate for 16-18 hours, and then replace with new complete medium for culture.
  • the total culture time is 48 hours.
  • step 2 digest and collect cells with trypsin, extract genomic DNA, and use a primer pair consisting of MSTN-F896 and MSTN-R1351 (three sets of MSTN) for PCR amplification, or use FNDC5-F209 and FNDC5- A primer pair consisting of R718 (three sets of FNDC5) was subjected to PCR amplification and then electrophoresis.
  • the results of the three groups of MSTN are shown in Figure 20.
  • the results of the three groups of FNDC5 are shown in Figure 21.
  • 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 present invention can be used to obtain diabetic pig models by gene editing methods for drug screening, drug efficacy testing, disease pathology, gene therapy and cell therapy, etc., and can provide effective experimental data for further clinical applications and cure humans in the future Diabetes lays a solid foundation.
  • the invention lays a solid foundation for the preparation of the diabetic pig model, and has great application value for the research and development of diabetic drugs.

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Abstract

提供了一种用于制备IRS基因缺陷的糖尿病克隆猪核供体细胞的CRISPR系统,其中使用的sgRNA组合由sgRNA IRS1-1、sgRNA IRS1-3、sgRNA IRS2-2和sgRNA IRS2-3组成。该系统用于制备糖尿病动物细胞模型及糖尿病动物模型。

Description

一种用于制备IRS基因缺陷的糖尿病克隆猪核供体细胞的CRISPR系统及其应用 技术领域
本发明涉及一种用于制备IRS基因缺陷的糖尿病克隆猪核供体细胞的CRISPR系统及其应用。
背景技术
糖尿病(diabetes mellitus,DM)是一种代谢性疾病，其特征是患者的血糖长期高于标准值。糖尿病又可分为1型糖尿病和2型糖尿病，其中2型糖尿病患者占总糖尿病患者的90％以上。1型糖尿病又称胰岛素依赖型糖尿病，通常是由于胰岛β-细胞损害导致患者自身不能分泌胰岛素引起的，其主要临床表现为多食、多饮、多尿及体重减少的“三多一少”症状；2型糖尿病又称非胰岛素依赖型糖尿病，通常是由于胰岛素调控葡萄糖代谢能力的下降伴随胰岛β细胞功能缺陷所导致的胰岛素分泌减少引起的，其主要临床表现为发病前肥胖，疲乏无力，若得不到及时诊断，体重又会逐渐下降。不论是哪一种糖尿病，如果不进行及时的治疗，均会引发心血管疾病、中风、慢性肾脏病、糖尿病足、以及视网膜病变等一系列并发症，对患者造成危害。据国际糖尿病联盟2017年调查数据显示，目前全球约有4.25亿人患有糖尿病，成人发病率约为8.8％，其中约有400万人死于糖尿病及其并发症。糖尿病目前已经成为仅次于心脑血管疾病、肿瘤之后第三大威胁人类健康的主要疾病。中国作为全球糖尿病患者最多的国家，2017年患病人数就已高达1.144亿人，成人发病率高达10.9％，已远超世界平均水平。而在1980年，中国的糖尿病患病率仅为0.67％。目前在我国糖尿病患者中，1型糖尿病仅占5％左右，从世界范围来说，中国甚至是世界上1型糖尿病患病率最低的国家，但却是2型糖尿病的高发病国家。
遗传上，1型糖尿病和2型糖尿病都不是仅仅由单基因控制，而是由多基因和环境共同作用影响的复杂疾病。众多研究表明至少有超过20个不同的基因组区域与1型糖尿病密切关联；在不同人群的全基因组扫描研究中，也发现了许多不同的与2型糖尿病密切关联的基因组区域。此外，众多证据显示1型和2型糖尿病的易感性及抗性都有明显的家族聚集倾向。目前，已鉴别到的1型糖尿病的主效致病基因为位于6号染色体的人类白细胞抗原(human leukocyte antigen,HLA)基因，该基因能够解释1型糖尿病40％-50％的遗传易感性。而2型糖尿病在不同人群中易感机理差别较大，目前认为编码胰岛素受体底物蛋白基因IRS-1和IRS-2基因缺陷与2型糖尿病密切相关。
目前尚无根治糖尿病的方法，总的治疗原则是通过改变生活方式，控制饮食，并配合一定的药物，以达到控制血糖、预防并发症的目的。对于1型糖尿病，通常患者采用注射胰岛素的手段来达到降低血糖的目的；对于2型糖尿病，通常患者采取口服降糖药的手段来达到降低血糖的目的。近年来，基因疗法的迅速发展为糖尿病的治疗带来了新的可能。
目前，在研究糖尿病的疾病动物模型上以小鼠模型为主，其又可分为实验诱导 糖尿病小鼠模型和自发性糖尿病小鼠模型两种。近年来，已有研究通过向β细胞-毒素诱导的糖尿病小鼠和自身免疫性非肥胖糖尿病小鼠体内注入带有Pdx1和MafA表达的腺病毒，将α细胞重编辑为功能性β细胞，并暂时恢复了小鼠的血糖水平。
猪作为大动物，是人类主要的肉食供应动物，其易于规模化繁殖饲养，且猪体型大小、生理功能及解剖结构等均与人相近，是理想的人类疾病模型动物。目前，已有通过四氧嘧啶或链脲霉素诱导的2型糖尿病猪模型，但实验诱导的动物疾病模型并不能完全模拟人类的真实疾病，而自发性疾病动物模型由于个体样本数量少很难实现大规模研究。
发明公开
本发明的目的是提供一种用于制备IRS基因缺陷的糖尿病克隆猪核供体细胞的CRISPR系统及其应用。
本发明提供了sgRNA组合，为如下(a1)、(a2)或(a3)：
(a1)由sgRNA IRS1-1、sgRNA IRS1-3、sgRNA IRS2-2和sgRNA IRS2-3组成的sgRNA组合；
(a2)由sgRNA IRS1-1和sgRNA IRS1-3组成的sgRNA组合；
(a3)由sgRNA IRS2-2和sgRNA IRS2-3组成的sgRNA组合。
本发明提供了质粒组合，为如下(b1)、(b2)或(b3)：
(b1)由质粒IRS1-1、质粒IRS1-3、质粒IRS2-2和质粒IRS2-3组成的质粒组合；
(b2)由质粒IRS1-1和质粒IRS1-3组成的质粒组合；
(b3)由质粒IRS2-2和质粒IRS2-3组成的质粒组合。
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。所述质粒组合的质粒的总摩尔数与质粒pKG-GE3的摩尔数的配比具体可为3：1。
以上任一所述试剂盒的用途为如下(c1)或(c2)或(c3)：(c1)制备重组细胞；(c2)制备糖尿病动物模型；(c3)制备糖尿病动物细胞模型。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。制备糖尿病动物模型时，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，即为糖尿病动物模型。还可以用糖尿病动物模型制备糖尿病动物细胞模型，即分离糖尿病动物模型的相应细胞，作为糖尿病动物细胞模型。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。应用时，sgRNA质粒总摩尔数和Cas质粒(具 体可为质粒pKG-GE3)摩尔数的配比具体可为3：1。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胰岛素受体底物基因缺陷的细胞。
以上任一所述重组细胞为胰岛素受体底物1基因缺陷的细胞。
以上任一所述重组细胞为胰岛素受体底物2基因缺陷的细胞。
以上任一所述重组细胞为胰岛素受体底物1基因和胰岛素受体底物2基因均缺陷的细胞。
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糖尿病动物模型中的应用。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糖尿病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，即为糖尿病动物模型。所述重组细胞为猪重组细胞。制备所述重组细胞时，sgRNA质粒总摩尔数和Cas质粒(具体可为质粒pKG-GE3)摩尔数的配比具体可为3：1。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。可以用糖尿病动物模型制备糖尿病动物细胞模型，即分离糖尿病动物模型的相应细胞，作为糖尿病动物细胞模型。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。
本发明还保护一种制备重组细胞的方法，包括如下步骤：将质粒IRS1-1、质粒IRS1-3、质粒IRS2-2、质粒IRS2-3和质粒pKG-GE3共转染猪细胞，得到胰岛素受体底物1基因发生突变且胰岛素受体底物2基因发生突变的重组细胞。质粒IRS1-1、质粒IRS1-3、质粒IRS2-2和质粒IRS2-3的总摩尔数与质粒pKG-GE3的摩尔数的配比为3：1。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。所述重组细胞具体可为胰岛素受体底物1基因发生杂合突变(对应基因型为杂合突变型)且胰岛素受体底物2基因发生特定突变的重组细胞；所述特定突变为纯合突变(对应的基因型为纯合突变型)或双等位突变(对应的基因型为双等位突变型)。
本发明还保护一种制备重组细胞的方法，包括如下步骤：将质粒IRS1-1、质粒IRS1-3和质粒pKG-GE3共转染猪细胞，得到胰岛素受体底物1基因发生突变的重组细胞。质粒IRS1-1和质粒IRS1-3的总摩尔数与质粒pKG-GE3的摩尔数的配比为3：1。所述突变为杂合突变、纯合突变或双等位突变。所述猪细胞为猪成纤维细胞。所述猪细胞为猪原代成纤维细胞。所述猪具体可为从江香猪。
本发明还保护一种制备重组细胞的方法，包括如下步骤：将质粒IRS2-2、质粒IRS2-3和质粒pKG-GE3共转染猪细胞，得到胰岛素受体底物2基因发生突变的重组细胞。质粒IRS2-2和质粒IRS2-3的总摩尔数与质粒pKG-GE3的摩尔数的配比为3：1。所述突变为杂合突变、纯合突变或双等位突变。所述猪细胞为猪成纤维细胞。 所述猪细胞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方法制备得到的重组细胞。具体来说，所述重组细胞可为表3、表4或表5中任一所述的重组细胞。具体来说，所述重组细胞可为如下任一：表3中编号为3、10、16、22、4、5、6、7、17或24的单克隆细胞。具体来说，所述重组细胞可为如下任一：表4中编号为25、28、30、35、47、33、37、41或48的单克隆细胞。具体来说，所述重组细胞可为如下任一：表5中编号为51、66或70的单克隆细胞。
本发明还保护所述重组细胞在制备糖尿病动物模型中的应用。本发明还保护所述重组细胞在制备糖尿病动物细胞模型中的应用。制备糖尿病动物模型时，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，即为糖尿病动物模型。可以用糖尿病动物模型制备糖尿病动物细胞模型，即分离糖尿病动物模型的相应细胞，作为糖尿病动物细胞模型。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。
以上任一所述糖尿病具体可为2型糖尿病。
所述sgRNA IRS1-1的靶序列结合区如SEQ ID NO：8中第1-20位核苷酸所示。所述sgRNA IRS1-1具体如SEQ ID NO：8所示。
所述sgRNA IRS1-3的靶序列结合区如SEQ ID NO：10中第1-20位核苷酸所示。所述sgRNA IRS1-3具体如SEQ ID NO：10所示。
所述sgRNA IRS2-2的靶序列结合区如SEQ ID NO：14中第1-20位核苷酸所示。所述sgRNA IRS2-2具体如SEQ ID NO：14所示。
所述sgRNA IRS2-3的靶序列结合区如SEQ ID NO：15中第1-20位核苷酸所示。所述sgRNA IRS2-3具体如SEQ ID NO：15所示。
所述质粒IRS1-1转录得到sgRNA IRS1-1。
所述质粒IRS1-3转录得到sgRNA IRS1-3。
所述质粒IRS2-2转录得到sgRNA IRS2-2。
所述质粒IRS2-3转录得到sgRNA IRS2-3。
具体来说，所述质粒IRS1-1是借助限制性内切酶BbsI将sgRNA IRS1-1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IRS1-3是借助限制性内切酶BbsI将sgRNA IRS1-3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IRS2-2质粒是借助限制性内切酶BbsI将sgRNA IRS2-2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IRS2-3是借助限制性内切酶BbsI将sgRNA IRS2-3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IRS1基因信息：GeneID为100512686，Sus scrofa。猪IRS1基因编码insulin receptor substrate 1。猪IRS1基因编码的蛋白质如SEQ ID NO：4所示。基因组DNA中，猪IRS1基因具有2个外显子，第一个外显子如SEQ ID NO：6所示，第二个外显子如SEQ ID NO：7所示。猪IRS1基因的开放阅读框如SEQ ID NO：6中第1-3726位核苷酸所示。IRS1基因为编码SEQ ID NO：4所示蛋白质的基因。IRS1基因为具有SEQ ID NO：6所示DNA分子的基因。IRS1基因为猪IRS1基因。
猪IRS2基因信息：GeneID为110255858，Sus scrofa。猪IRS2基因编码insulin receptor substrate 2。猪IRS2基因编码的蛋白质如SEQ ID NO：11所示。基因组DNA中，猪IRS2基因具有2个外显子，第一个外显子的编码区如SEQ ID NO：12中第1-4006位核苷酸所示，第二个外显子的编码区如SEQ ID NO：12中第4007-4011位核苷酸所示。猪IRS2基因的开放阅读框如SEQ ID NO：12所示。IRS2基因为编码SEQ ID NO：11所示蛋白质的基因。IRS2基因为具有SEQ ID NO：12所示DNA分子的基因。IRS2基因为猪IRS2基因。
与现有技术相比，本发明至少具有如下有益效果：
(1)对每个靶基因，本发明采用双gRNA组合进行突变，与采用单gRNA相比，可有效降低非移码突变的产生，并可直接用PCR来检测基因编辑效率。如果用单gRNA对靶基因进行突变，在DNA的非同源末端连接(NHEJ)随机修复中，会有1/3的概率产生碱基的非移码突变，而非移码突变很可能不能破坏靶基因的功能，达不到使靶基因失活的预期目标。而用双gRNA对靶基因进行切割突变时，可使靶基因去除掉一个片段，通过设计去除非3倍数的碱基片段，可有效产生靶基因的片段缺失移码突变。同时也可以直接通过PCR手段来检测缺失片段的基因编辑产物，通过基因编辑产物与野生型产物(即未编辑产物)的比率可直接估算基因编辑的效率。另外，双gRNA除可造成理论片段缺失，还存在单个gRNA的分别单独切割情况，从而大大增加基因突变的效率。
(2)gRNA载体和cas9载体不是按常规的1：1摩尔数比，而是按3：1的摩尔数比。两个gRNA质粒与Cas9质粒最适用量为摩尔比1.5：1.5：1，质粒实际用量为0.46ug+0.46ug+1.08ug。针对最终起作用的gRNA：cas9蛋白复合物来讲，gRNA载体转录出gRNA的时间要比cas9蛋白形成的时间早，而且转录出来的gRNA降解速度很快，因此如果在DNA载体水平，摩尔数比按1：1的话，由于gRNA的早转录及降解，最终会使cas9蛋白的摩尔数多于未降解的gRNA。经过实验比对，发现3:1或4:1要比1:1的gRNA:cas9载体摩尔比编辑效率更高。因此，本发明优选采用了3:1的gRNA:cas9的载体摩尔比。
(3)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。目前为止，仅有自发突变型小鼠糖尿病疾病模型被选育出，未有任何大动物糖尿病疾病模型被成功研发。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。
(4)采用本发明改造的cas9高效表达载体进行基因编辑,编辑效率比原载体提高3～4倍。
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的步骤一中以8只猪的基因组DNA为模板采用引物IRS1-GT-F412/IRS1-GT-R1220组成的引物对进行PCR扩增后的电泳图。
图6为实施例2的步骤三中各种具有粘性末端的双链DNA分子。
图7为实施例2的步骤四中以基因组DNA采用IRS1-F583和IRS1-R961组成的引物对进行PCR扩增后的电泳图。
图8为以8只猪的基因组DNA为模板采用引物引物IRS2-GT-nF848/IRS2-GT-nR1710组成的引物对进行PCR扩增后的电泳图。
图9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10为实施例3的步骤四中以基因组DNA采用IRS2-GT-nF848和IRS2-GT-nR1502组成的引物对进行PCR扩增后的电泳图。
图11为实施例4中第一组得到的细胞的电泳图。
图12为实施例4中第二组得到的细胞的电泳图。
图13为实施例4中第三组得到的细胞的电泳图(IRS1-F583和IRS1-R961组成的引物对)。
图14为实施例4中第三组得到的细胞的电泳图(IRS2-GT-nF848和IRS2-GT-nR1502组成的引物对)。
图15为IRS1-3的测序峰图。
图16为IRS1-4的测序峰图。
图17为IRS2-25的测序峰图。
图18为IRS2-26的测序峰图。
图19为实施例5的步骤二中以基因组DNA为模板采用MSTN-F896和MSTN-R1351组成的引物对进行PCR扩增后的电泳图。
图20为实施例5的步骤三中MSTN三组的电泳图。
图21为实施例5的步骤三中FNDC5三组的电泳图。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。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5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、IRS1基因敲除的靶点组合筛选
一、IRS1基因敲除预设靶点及邻近基因组序列保守性分析
猪IRS1基因信息：编码insulin receptor substrate 1；位于15号染色体；GeneID为100512686，Sus scrofa。猪IRS1基因编码的蛋白质如SEQ ID NO：4所示。基因组DNA中，猪IRS1基因具有2个外显子，第一个外显子如SEQ ID NO：6所示，第二个外显子如SEQ ID NO：7所示。基因组DNA中，位于猪IRS1基因第一个外显子上游的部分核苷酸如SEQ ID NO：5所示。猪IRS1基因的开放阅读框位于第一个外显子，如SEQ ID NO：6中第1-3726位核苷酸所示。
分别以8只猪的基因组DNA为模板，采用引物IRS1-GT-F412/IRS1-GT-R1220组成的引物对进行PCR扩增，然后进行电泳，见图5。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IRS1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：IRS1-F583和IRS1-R961。
IRS1-GT-F412：5’-GCATGAAACGCCAGTAAACTCCG-3’；
IRS1-GT-R1220：5’-CGAAACTGATGGTCTTGCTGGTC-3’。
IRS1-F583：5’-CCACCCGGTTGTTTTTCGGCG-3’；
IRS1-R961：5’-CTGGTACCAGCTGTCCTGTTCG-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3个靶点。
3个靶点分别如下：
sgRNA IRS1-1靶点：5’-CTCGTAGTACTCGAGGCGCG-3’；
sgRNA IRS1-2靶点：5’-ATGTTGAAGCAGCTCTCCAG-3’；
sgRNA IRS1-3靶点：5’-GCTGCTTCAACATCAACAAG-3’。
各靶点组合及造成的理论缺失见表1。
表1 靶点组合及造成的理论缺失
5’端靶点序列 方向 编号 3’端靶点序列 方向 编号 缺失长度bp
CTCGTAGTACTCGAGGCGCG - 1 ATGTTGAAGCAGCTCTCCAG - 2 71
CTCGTAGTACTCGAGGCGCG - 1 GCTGCTTCAACATCAACAAG + 3 92
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IRS1-1S和IRS1-1A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图6A)。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(IRS1-1)。质粒pKG-U6gRNA(IRS1-1)表达SEQ ID NO：8所示的sgRNA IRS1-1。
SEQ ID NO：8：
Figure PCTCN2020124633-appb-000001
分别合成IRS1-2S和IRS1-2A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图6B)。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(IRS1-2)。质粒pKG-U6gRNA(IRS1-2)表达SEQ ID NO：9所示的sgRNA IRS1-2。
SEQ ID NO：9：
Figure PCTCN2020124633-appb-000002
分别合成IRS1-3S和IRS1-3A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图6C)。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(IRS1-3)。质粒pKG-U6gRNA(IRS1-3)表达SEQ ID NO：10所示的sgRNA IRS1-3。
SEQ ID NO：10：
Figure PCTCN2020124633-appb-000003
IRS1-1S：5’-caccgCTCGTAGTACTCGAGGCGCG-3’；
IRS1-1A：5’-aaacCGCGCCTCGAGTACTACGAGc-3’。
IRS1-2S：5’-caccgATGTTGAAGCAGCTCTCCAG-3’；
IRS1-2A：5’-aaacCTGGAGAGCTGCTTCAACATc-3’。
IRS1-3S：5’-caccGCTGCTTCAACATCAACAAG-3’；
IRS1-3A：5’-aaacCTTGTTGATGTTGAAGCAGC-3’。
IRS1-1S、IRS1-1A、IRS1-2S、IRS1-2A、IRS1-3S、IRS1-3A，均为单链DNA分子。
四、不同靶点组合的编辑效率比较
1、共转染
第一组：将质粒pKG-U6gRNA(IRS1-1)、质粒pKG-U6gRNA(IRS1-2)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(IRS1-1)：0.46μg质粒pKG-U6gRNA(IRS1-2)：1.08μg质粒pKG-GE3。
第二组：将质粒pKG-U6gRNA(IRS1-1)、质粒pKG-U6gRNA(IRS1-3)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(IRS1-1)：0.46μg质粒pKG-U6gRNA(IRS1-3)：1.08μg质粒pKG-GE3。
第三组：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IRS1-F583和IRS1-R961组成的引物对进行PCR扩增，然后进行电泳。结果见图7。较大条带为野生型条带(WT)，较小条带为突变条带(MT)，突变条带相对野生型条带越亮，则突变效率越高。
基因缺失突变效率＝(MT灰度/MT条带bp数)/(WT灰度/WT条带bp数+MT灰度/MT条带bp数)×100％。第一组基因缺失突变效率为57％，第二组基因缺失突变效率为65％，第三组基因缺失突变效率为0％。
结果表明，第二组效果最好，sgRNA IRS1-1和sgRNA IRS1-3为最优组合。
实施例3、IRS2基因敲除的靶点组合筛选
一、IRS2基因敲除预设靶点外显子及邻近基因组序列保守性分析
猪IRS2基因信息：编码insulin receptor substrate 2；位于11号染色体；GeneID为110255858，Sus scrofa。猪IRS2基因编码的蛋白质如SEQ ID NO：11所示。基因组DNA中，猪IRS2基因具有2个外显子，第一个外显子的编码区如SEQ ID NO：12中第1-4006位核苷酸所示，第二个外显子的编码区如SEQ ID NO：12中第4007-4011位核苷酸所示。
分别以8只猪的基因组DNA为模板，采用引物IRS2-GT-nF848/IRS2-GT-nR1710组成的引物对进行PCR扩增，然后进行电泳，见图8。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IRS2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：IRS2-GT-nF848和IRS2-GT-nR1502。
IRS2-GT-nF848：5’-AGAACATCCACGAGACCATCCTG-3’；
IRS2-GT-nR1710：5’-TCTCAGCCCTCTATCCAAGTCCT-3’；
IRS2-GT-nR1502：5’-TCATCCAGGGACATAAAGCCAGG-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 IRS2-1靶点：5’-GACGACTGGCTCTTGCTGCG-3’；
sgRNA IRS2-2靶点：5’-GGTTGACCAGGTGGTGGTGG-3’；
sgRNA IRS2-3靶点：5’-CACGAGCTGCACTTGGCCGC-3’；
sgRNA IRS2-4靶点：5’-AACCCGGCACGAGCTGCACT-3’。
各靶点组合及造成的理论缺失见表2。
表2 靶点组合及造成的理论缺失表
5’端靶点序列 方向 编号 3’端靶点序列 方向 编号 缺失长度bp
GACGACTGGCTCTTGCTGCG - 1 GGTTGACCAGGTGGTGGTGG - 2 65
GACGACTGGCTCTTGCTGCG - 1 AACCCGGCACGAGCTGCACT - 4 157
GGTTGACCAGGTGGTGGTGG - 2 CACGAGCTGCACTTGGCCGC - 3 85
GGTTGACCAGGTGGTGGTGG - 2 AACCCGGCACGAGCTGCACT - 4 92
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IRS2-1S和IRS2-1A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图9A)。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(IRS2-1)。质粒pKG-U6gRNA(IRS2-1)表达SEQ ID NO：13所示的sgRNA IRS2-1。
SEQ ID NO：13：
Figure PCTCN2020124633-appb-000004
分别合成IRS2-2S和IRS2-2A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图9B)。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(IRS2-2)。质粒pKG-U6gRNA(IRS2-2)表达SEQ ID NO：14所示的sgRNA IRS2-2。
SEQ ID NO：14：
Figure PCTCN2020124633-appb-000005
分别合成IRS2-3S和IRS2-3A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图9C)。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(IRS2-3)。质粒pKG-U6gRNA(IRS2-3)表达SEQ ID NO：15所示的sgRNA IRS2-3。
SEQ ID NO：15：
Figure PCTCN2020124633-appb-000006
分别合成IRS2-4S和IRS2-4A，然后混合并进行退火，得到具有粘性末端的双链DNA分子(图9D)。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(IRS2-4)。质粒pKG-U6gRNA(IRS2-4)表达SEQ ID NO：16所示的sgRNA IRS2-4。
SEQ ID NO：16：
Figure PCTCN2020124633-appb-000007
IRS2-1S：5’-caccGACGACTGGCTCTTGCTGCG-3’；
IRS2-1A：5’-aaacCGCAGCAAGAGCCAGTCGTC-3’。
IRS2-2S：5’-caccGGTTGACCAGGTGGTGGTGG-3’；
IRS2-2A：5’-aaacCCACCACCACCTGGTCAACC-3’。
IRS2-3S：5’-caccgCACGAGCTGCACTTGGCCGC-3’；
IRS2-3A：5’-aaacGCGGCCAAGTGCAGCTCGTGc-3’。
IRS2-4S：5’-caccgAACCCGGCACGAGCTGCACT-3’；
IRS2-4A：5’-aaacAGTGCAGCTCGTGCCGGGTTc-3’。
IRS2-1S、IRS2-1A、IRS2-2S、IRS2-2A、IRS2-3S、IRS2-3A、IRS2-4S、IRS2-4A，均为单链DNA分子。
四、不同靶点组合的编辑效率比较
1、共转染
第一组：将质粒pKG-U6gRNA(IRS2-1)、质粒pKG-U6gRNA(IRS2-2)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(IRS2-1)：0.46μg质粒pKG-U6gRNA(IRS2-2)：1.08μg质粒pKG-GE3。
第二组：将质粒pKG-U6gRNA(IRS2-1)、质粒pKG-U6gRNA(IRS2-4)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(IRS2-1)：0.46μg质粒pKG-U6gRNA(IRS2-4)：1.08μg质粒pKG-GE3。
第三组：将质粒pKG-U6gRNA(IRS2-2)、质粒pKG-U6gRNA(IRS2-3)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(IRS2-2)：0.46μg质粒pKG-U6gRNA(IRS2-3)：1.08μg质粒pKG-GE3。
第四组：将质粒pKG-U6gRNA(IRS2-2)、质粒pKG-U6gRNA(IRS2-4)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(IRS2-2)：0.46μg质粒pKG-U6gRNA(IRS2-4)：1.08μg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IRS2-GT-nF848和IRS2-GT-nR1502组成的引物对进行PCR扩增，然后进行电泳。结果见图10。较大条带为野生型条带(WT)，较小条带为突变条带(MT)，突变条带相对野生型条带越亮，则突变效率越高。
基因缺失突变效率＝(MT灰度/MT条带bp数)/(WT灰度/WT条带bp数+MT灰度/MT条带bp数)×100％。第一组基因缺失突变效率为50％，第二组基因缺失突变效率为未检出，第三组基因缺失突变效率为91％。第四组基因缺失突变效率为82％，第五组(阴性对照组)基因缺失突变效率为0％。
结果表明，第三组效果最好，sgRNA IRS2-2和sgRNA IRS2-3为最优组合。
实施例4、制备IRS1基因敲除单克隆细胞、IRS2基因敲除单克隆细胞、IRS1和IRS2基因联合敲除的单克隆细胞
1、共转染
第一组：将质粒pKG-U6gRNA(IRS1-1)、质粒pKG-U6gRNA(IRS1-3)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(IRS1-1)：0.46μg质粒pKG-U6gRNA(IRS1-3)：1.08μg质粒pKG-GE3。
第二组：将质粒pKG-U6gRNA(IRS2-2)、质粒pKG-U6gRNA(IRS2-3)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(IRS2-2)：0.46μg质粒pKG-U6gRNA(IRS2-3)：1.08μg质粒pKG-GE3。
第三组：将质粒pKG-U6gRNA(IRS1-1)、质粒pKG-U6gRNA(IRS1-3)、质粒pKG-U6gRNA(IRS2-2)、质粒pKG-U6gRNA(IRS2-3)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：0.23μg质粒pKG-U6gRNA(IRS1-1)：0.23μg质粒pKG-U6gRNA(IRS1-3)：0.23μg质粒pKG-U6gRNA(IRS2-2)：0.23μg质粒pKG-U6gRNA(IRS2-3)：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，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(第一组得到的细胞采用IRS1-F583和IRS1-R961组成的引物对进行PCR扩增，第二组得到的细胞采用IRS2-GT-nF848和IRS2-GT-nR1502组成的引物对进行PCR扩增，第三组得到的细胞分别采用上述两个引物对进行PCR扩增)，然后进行电泳。将猪原代成纤维细胞作为野生型对照。
第一组得到的细胞的电泳图见图11。第二组得到的细胞的电泳图见图12。第三组得到的细胞(IRS1-F583和IRS1-R961组成的引物对)的电泳图见图13。第三组得到的细胞(IRS2-GT-nF848和IRS2-GT-nR1502组成的引物对)的电泳图见图 14。图11至图14中，泳道编号与单克隆细胞编号一致。
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纯合突变型；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。
第一组的结果见表3。编号为3、10、16、22的单克隆细胞的基因型为纯合突变型。编号为4、5、6、7、17、24的单克隆细胞的基因型为双等位突变型。编号为8、11、13、15、19、20、23的单克隆细胞的基因型为杂合型。编号为1、2、9、12、14、18、21的单克隆细胞的基因型为纯合野生型。第一组得到IRS1基因编辑单克隆细胞的比率为17/24。示例性的，IRS1-3的测序峰图见图15，IRS1-4的测序峰图见图16。
第二组的结果见表4。编号为25、28、30、35、47的单克隆细胞的基因型为纯合突变型。编号为33、37、41、48的单克隆细胞的基因型为双等位突变型。编号为26、34、36、38、39、42、43、45的单克隆细胞的基因型为杂合型。编号为27、29、31、32、40、44、46的单克隆细胞的基因型为纯合野生型。第二组得到IRS2基因编辑单克隆细胞的比率为17/24。示例性的，IRS2-25的测序峰图见图17，IRS2-26的测序峰图见图18。
第三组的结果见表5。基于IRS1基因的基因型和基于IRS2基因的基因型均为纯合突变型的单克隆细胞的编号为56、63。基于IRS1基因的基因型和基于IRS2基因的基因型均为双等位突变型的单克隆细胞的编号为54、59。基于IRS1基因的基因型为杂合型且基于IRS2基因的基因型为纯合突变/双等位突变的单克隆细胞的编号为51、66、70。
表3
Figure PCTCN2020124633-appb-000008
Figure PCTCN2020124633-appb-000009
表4
Figure PCTCN2020124633-appb-000010
Figure PCTCN2020124633-appb-000011
表5
Figure PCTCN2020124633-appb-000012
Figure PCTCN2020124633-appb-000013
实施例5、质粒配比优化以及质粒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’。
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、质粒配比优化
第一组：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：0.22μg质粒pKG-U6gRNA(MSTN-1)：0.22μg质粒pKG-U6gRNA(MSTN-2)：1.56μg质粒pKG-GE3。即质粒pKG-U6gRNA(MSTN-1)、质粒pKG-U6gRNA(MSTN-2)和质粒pKG-GE3的摩尔配比依次为：0.5：0.5：1。
第二组：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：0.36μg质粒pKG-U6gRNA(MSTN-1)：0.36μg质粒pKG-U6gRNA(MSTN-2)：1.27μg质粒pKG-GE3。即质粒pKG-U6gRNA(MSTN-1)、质粒pKG-U6gRNA(MSTN-2)和质粒pKG-GE3的摩尔配比依次为：1：1：1。
第三组：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：0.46μg质粒pKG-U6gRNA(MSTN-1)：0.46μg质粒pKG-U6gRNA(MSTN-2)：1.08μg质粒pKG-GE3。即质粒pKG-U6gRNA(MSTN-1)、质粒pKG-U6gRNA(MSTN-2)和质粒pKG-GE3的摩尔配比依次为1.5：1.5：1。
第四组：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：0.53μg质粒pKG-U6gRNA(MSTN-1)：0.53μg质粒pKG-U6gRNA(MSTN-2)：0.93μg质粒pKG-GE3。即质粒pKG-U6gRNA(MSTN-1)、质粒pKG-U6gRNA(MSTN-2)和质粒pKG-GE3的摩尔配比依次为：2：2：1.
第五组：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：1μg质粒pKG-U6gRNA(MSTN-1)：1μg质粒pKG-U6gRNA(MSTN-2)。
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组成的引物对进行PCR扩增，然后进行电泳。
电泳结果见图19。456bp条带为野生型条带(WT)，329bp左右(条带456bp理论缺失127bp)为缺失突变条带(MT)。
基因缺失突变效率＝(MT灰度/MT条带bp数)/(WT灰度/WT条带bp数+MT灰度/MT条带bp数)×100％。第一组基因缺失突变效率为28.6％，第二组基因缺失突变效率为77.8％，第三组基因缺失突变效率为86.8％，第四组基因缺失突变效率为81.5％。第三组的基因编辑效率最高，确定两个gRNA质粒与Cas9质粒最适用量为摩尔比1.5：1.5：1，质粒实际用量为0.46μg：0.46μg：1.08μg。
三、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20。FNDC5的三组的结果见图21。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，为质粒pX330的3～4倍。
工业应用
本发明可用于通过基因编辑手段获得糖尿病猪模型，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，为今后治愈人类糖尿病打下坚实基础。本发明为糖尿病猪模型的制备奠定了坚实的基础，对于糖尿病药物的研发具有重大应用价值。

Claims (17)

  1. sgRNA组合，为如下(a1)、(a2)或(a3)：
    (a1)由sgRNA IRS1-1、sgRNA IRS1-3、sgRNA IRS2-2和sgRNA IRS2-3组成的sgRNA组合；
    (a2)由sgRNA IRS1-1和sgRNA IRS1-3组成的sgRNA组合；
    (a3)由sgRNA IRS2-2和sgRNA IRS2-3组成的sgRNA组合；
    所述sgRNA IRS1-1的靶序列结合区如SEQ ID NO：8中第1-20位核苷酸所示；
    所述sgRNA IRS1-3的靶序列结合区如SEQ ID NO：10中第1-20位核苷酸所示；
    所述sgRNA IRS2-2的靶序列结合区如SEQ ID NO：14中第1-20位核苷酸所示；
    所述sgRNA IRS2-3的靶序列结合区如SEQ ID NO：15中第1-20位核苷酸所示。
  2. 质粒组合，为如下(b1)、(b2)或(b3)：
    (b1)由质粒IRS1-1、质粒IRS1-3、质粒IRS2-2和质粒IRS2-3组成的质粒组合；
    (b2)由质粒IRS1-1和质粒IRS1-3组成的质粒组合；
    (b3)由质粒IRS2-2和质粒IRS2-3组成的质粒组合；
    所述质粒IRS1-1转录得到sgRNA IRS1-1；所述质粒IRS1-3转录得到sgRNA IRS1-3；所述质粒IRS2-2转录得到sgRNA IRS2-2；所述质粒IRS2-3转录得到sgRNA IRS2-3；
    所述sgRNA IRS1-1的靶序列结合区如SEQ ID NO：8中第1-20位核苷酸所示；
    所述sgRNA IRS1-3的靶序列结合区如SEQ ID NO：10中第1-20位核苷酸所示；
    所述sgRNA IRS2-2的靶序列结合区如SEQ ID NO：14中第1-20位核苷酸所示；
    所述sgRNA IRS2-3的靶序列结合区如SEQ ID NO：15中第1-20位核苷酸所示。
  3. 一种试剂盒，包括权利要求1所述的sgRNA组合；所述试剂盒的用途为如下(c1)或(c2)或(c3)：(c1)制备重组细胞；(c2)制备糖尿病动物模型；(c3)制备糖尿病动物细胞模型。
  4. 一种试剂盒，包括权利要求2所述的质粒组合；所述试剂盒的用途为如下(c1)或(c2)或(c3)：(c1)制备重组细胞；(c2)制备糖尿病动物模型；(c3)制备糖尿病动物细胞模型。
  5. 如权利要求4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。
  6. 权利要求1所述sgRNA组合在制备试剂盒中的应用；所述试剂盒的用途为如下(c1)或(c2)或(c3)：(c1)制备重组细胞；(c2)制备糖尿病动物模型； (c3)制备糖尿病动物细胞模型。
  7. 权利要求2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(c1)或(c2)或(c3)：(c1)制备重组细胞；(c2)制备糖尿病动物模型；(c3)制备糖尿病动物细胞模型。
  8. 权利要求2所述质粒组合和权利要求5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(c1)或(c2)或(c3)：(c1)制备重组细胞；(c2)制备糖尿病动物模型；(c3)制备糖尿病动物细胞模型。
  9. 权利要求1所述sgRNA组合、权利要求2所述质粒组合、权利要求3所述试剂盒、权利要求4所述试剂盒或权利要求5所述试剂盒在制备重组细胞中的应用。
  10. 权利要求1所述sgRNA组合、权利要求2所述质粒组合、权利要求3所述试剂盒、权利要求4所述试剂盒或权利要求5所述试剂盒在制备糖尿病动物模型中的应用。
  11. 权利要求1所述sgRNA组合、权利要求2所述质粒组合、权利要求3所述试剂盒、权利要求4所述试剂盒或权利要求5所述试剂盒在制备糖尿病动物细胞模型中的应用。
  12. 一种制备重组细胞的方法，包括如下步骤：将权利要求2中所述的质粒IRS1-1、质粒IRS1-3、质粒IRS2-2、质粒IRS2-3和权利要求5中所述的质粒pKG-GE3共转染猪细胞，得到胰岛素受体底物1基因发生突变且胰岛素受体底物2基因发生突变的重组细胞。
  13. 一种制备重组细胞的方法，包括如下步骤：将权利要求2中所述的质粒IRS1-1、质粒IRS1-3和权利要求5中所述的质粒pKG-GE3共转染猪细胞，得到胰岛素受体底物1基因发生突变的重组细胞。
  14. 一种制备重组细胞的方法，包括如下步骤：将权利要求2中所述的质粒IRS2-2、质粒IRS2-3和权利要求5中所述的质粒pKG-GE3共转染猪细胞，得到胰岛素受体底物2基因发生突变的重组细胞。
  15. 权利要求12至14中任一所述方法制备得到的重组细胞。
  16. 权利要求15所述重组细胞在制备糖尿病动物模型中的应用。
  17. 权利要求15所述重组细胞在制备糖尿病动物细胞模型中的应用。
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