Construction method of four-gene editing xenogenous organ transplantation donor pig

Abstract

The invention relates to a construction method of a four-gene editing xenogenic organ transplantation donor pig, and belongs to the technical field of animal biology. In a wild type pig fetal fibroblast cell line, a GGTA1 gene is knocked out by utilizing a CRISPR / Cas9 gene editing technology, three humanized genes of hCD39, hCD55 and hTBM are transferred, a GTKO / hCD39 / hCD55 / hTBM four-gene editing cloned pig is constructed in combination with a somatic cell nucleus transplantation technology, and four-gene editing xenogenic organ transplantation donor pigs are further obtained in batches through genotype identification and protein function phenotype analysis. According to the invention, the common problem of antigen-antibody mediated immunological rejection in a pig-human xenograft process is solved to a certain extent, and a foundation is laid for further research and development of donor pigs suitable for xenograft of organs / tissues such as heart, kidney, liver, skin and the like; the method has an important prospect for carrying out pre-clinical research on pig-non-human primate xenogeneic organ transplantation and clinical application of pig-human xenogeneic organ transplantation.

Description

Technical Field

[0001] The present invention belongs to the field of animal biotechnology, and in particular, relates to a method for constructing a four-gene edited xenotransplantation donor pig. Background Art

[0002] Organ transplantation is recognized as the main means of treating patients with organ failure in the international biomedical field. Every year, approximately 200,000 people around the world are waiting for organ transplantation, and the average organ supply-demand ratio is less than 1:20. The shortage of organ donors has become a major medical problem that is of concern and urgently needs to be solved in the field of public health around the world. Xenotransplantation has been recognized as an effective way to solve the global gap in supply and demand for human organ transplantation. When it comes to xenotransplantation, pigs are easy to raise and breed, their organ size matches that of humans, and their physiology and metabolism are very similar to humans. The possibility of human-pig co-morbidities is low, there are few ethical controversies, and genetic modification can be used to enhance the compatibility with donor organs. Therefore, pigs are recognized as the most suitable species for use as xenotransplantation donors, and pig-human xenotransplantation is hailed as the "next major biomedical revolution."

[0003] After nearly half a century of efforts and exploration, immune rejection is one of the main obstacles faced by xenotransplantation. At present, nearly 30 related genes that can effectively alleviate immunological incompatibility and overcome functional disorders have been screened. A variety of genetically modified donor pigs have also been developed and pig-non-human primate xenotransplantation experiments have been carried out. In December 2021, American scientists transplanted 10 gene-edited pig kidneys into brain-dead patients, and the kidneys functioned normally. In January 2023, the University of Maryland Medical Center transplanted a 10-gene-edited pig heart into a 57-year-old heart patient for the first time, and the patient survived for 2 months after the transplant. In August 2023, the University of Alabama at Birmingham transplanted 10 gene-edited pig kidneys into humans and they functioned normally for 1 week. In September 2023, the University of Maryland Medical Center successfully transplanted a 10-gene-edited pig heart into a patient with peripheral vascular disease and internal bleeding complications for the second time, and the patient survived for 6 weeks after the operation, marking the most critical step in the clinical application of pig-human xenotransplantation.

[0004] Although pigs have been recognized as a source of human organ transplantation, immunological incompatibility and physiological dysfunction remain major scientific issues and key technical challenges facing xenotransplantation. Therefore, the successful construction of a four-gene edited xenotransplantation donor pig will be of great significance for the research and promotion of xenotransplantation. Summary of the invention

[0005] In response to the problems in the above background technology, the present invention provides a method for constructing a four-gene edited xenotransplantation donor pig. This method targets the common problems of immunological incompatibility and complement regulation in pig-human xenotransplantation, and utilizes CRISPR / Cas9 gene editing combined with somatic cell cloning technology to construct a GTKO / hCD39 / hCD55 / hTBM four-gene edited xenotransplantation donor pig, which will lay the foundation for preclinical research on pig-non-human primate xenotransplantation and clinical application of pig-human xenotransplantation.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a GGTA1 gene targeting sgRNA expression vector, which utilizes CRISPR / Cas9 gene editing technology to construct a GGTA1 gene targeting sgRNA expression vector (GGTA1-sgRNA1 and GGTA1-sgRNA2), and the action site of the sgRNA expression vector is located in the 8th exon of the GGTA1 gene.

[0007] Furthermore, the nucleotide sequence of the GGTA1 gene targeting sgRNA expression vector, GGTA1-sgRNA1, is: 5′-GCTACAGGCCTGGTGGTACA-3′; the nucleotide sequence of GGTA1-sgRNA2 is: 5′-GATGCGCATGAAGACCATCG-3′.

[0008] Furthermore, the backbone vector to which the GGTA1 gene targeting sgRNA expression vector was connected was PX458-sgRNA-EGFP (Addgene no: 112220).

[0009] The second aspect of the present invention provides a recombinant plasmid for knocking out the GGTA1 gene in the pig genome, including: PX458-GGTA1-sgRNA1 and PX458-GGTA1-sgRNA2.

[0010] Furthermore, the nucleotide sequence of PX458-GGTA1-sgRNA1 is shown in SEQ ID NO:1, and the nucleotide sequence of PX458-GGTA1-sgRNA2 is shown in SEQ ID NO:2.

[0011] The third aspect of the present invention provides humanized gene expression vectors for hCD39, hCD55 and hTBM, including a recombinant plasmid for transferring hCD55 gene and a recombinant plasmid for transferring hCD39 and hTBM genes together.

[0012] Furthermore, the nucleotide sequence of the recombinant plasmid used to transfer the hCD55 gene is shown in SEQ ID NO:6.

[0013] Furthermore, the nucleotide sequence of the recombinant plasmid used for co-transferring hCD39 and hTBM genes is shown in SEQ ID NO:7.

[0014] The fourth aspect of the present invention provides a method for constructing a GTKO / hCD39 / hCD55 / hTBM four-gene edited porcine fibroblast cell line, wherein a GGTA1 gene targeting sgRNA expression vector or a recombinant plasmid for knocking out the GGTA1 gene in the porcine genome and a humanized gene expression vector are co-transfected into a porcine fetal fibroblast cell line under the action of a transposase, and the obtained four-gene edited positive cell monoclonal clone is the GTKO / hCD39 / hCD55 / hTBM four-gene edited porcine fibroblast cell line.

[0015] Furthermore, the blood type of the wild-type porcine fetal fibroblast cell line that has not undergone gene editing is type O. Type O blood has good compatibility and can reduce the immune rejection reaction caused by blood type incompatibility in xenotransplantation.

[0016] The fifth aspect of the present invention provides a method for constructing a four-gene edited xenotransplantation donor pig, the specific steps of which are: (1) Construction of GTKO gene knockout expression vector based on CRISPR / Cas9 gene editing system Targeting sgRNA1 and sgRNA2 were designed for exon 8 of the porcine GGTA1 gene and were connected to the backbone vector respectively to obtain the GGTA1 gene knockout sgRNA targeting vector. The nucleotide sequence of GGTA1-sgRNA1 is shown in SEQ ID NO: 1; the nucleotide sequence of GGTA1-sgRNA2 is shown in SEQ ID NO: 2.

[0017] (2) Construction of hCD39 / hCD55 / hTBM humanized gene expression vector based on piggyBac system Based on the piggyBac system, the humanized gene expression vectors of hCD39, hCD55, and hTBM were constructed using homologous recombination technology; Furthermore, the nucleotide sequence of hCD39 is shown in SEQ ID NO:3, the nucleotide sequence of hCD55 is shown in SEQ ID NO:4, and the nucleotide sequence of hTBM is shown in SEQ ID NO:5.

[0018] Furthermore, the nucleotide sequence of the recombinant plasmid used for transferring the hCD55 gene is shown in SEQ ID NO:6; the nucleotide sequence of the recombinant plasmid used for transferring the hCD39 and hTBM genes is shown in SEQ ID NO:7.

[0019] (3) Construction of GTKO / hCD39 / hCD55 / hTBM quadruple-gene edited porcine fibroblast cell line The recombinant plasmid used to knock out the GGTA1 gene in the pig genome and the hCD55 and hCD39 / hTBM humanized gene expression vectors were co-transfected into the wild-type porcine fetal fibroblast cell line, and the GTKO / hCD39 / hCD55 / hTBM four-gene edited porcine fibroblast cell line was obtained through single-cell clone genotype identification and screening.

[0020] (4) Somatic cell nuclear transfer and embryo transfer Using somatic cell nuclear transfer technology, the GTKO / hCD39 / hCD55 / hTBM four-gene edited porcine fibroblast cell line obtained by transfection screening is subjected to somatic cell nuclear transfer, and the fetus is removed during pregnancy to identify the genotype and isolate and culture the porcine fetal fibroblast cell line, or the four-gene edited cloned pig is obtained when it is born.

[0021] Furthermore, the above transfection method includes liposome transfection or electrofection; preferably electrofection.

[0022] (5) Genotyping and phenotypic analysis of cloned pigs The genomic DNA of cloned pigs was extracted, and their genotypes were identified by PCR, T7ENI, and Sanger sequencing techniques. The mRNA and protein expression levels of related genes in tissues, organs, and cells were detected by q-PCR technology, flow cytometry, tissue immunofluorescence technology, and Western blotting technology.

[0023] (6) Using continuous cloning technology, the four-gene edited porcine fetal fibroblast cell line or porcine fibroblast cell line obtained above is subjected to somatic cell nuclear transplantation, and the cloned embryos are transplanted into surrogate sows, so as to mass-produce GTKO / hCD39 / hCD55 / hTBM four-gene edited xenogeneic organ transplantation donor pigs.

[0024] The application of the above-mentioned targeting vector, recombinant plasmid or humanized gene expression vector, four-gene edited porcine fibroblast cell line or method for constructing four-gene edited xenotransplantation donor pig in pig-non-human primate xenotransplantation.

[0025] Knocking out the α-1,3-galactosyltransferase (GGTA1) gene can avoid the occurrence of hyperacute immune rejection reactions, and knocking out the porcine endogenous retrovirus (PERVs) gene can avoid cross-species infection of porcine viruses, solving the biosafety problem of xenotransplantation. However, immunological incompatibility and physiological functional disorders after xenotransplantation need more attention. Expressing human complement regulatory proteins (CD55) and thrombomodulin (CD39, TBM) in xenotransplantation donor pigs can correct the disorders of complement and coagulation caused by species incompatibility. Compared with GTKO single-gene edited pigs, GTKO / hCD55 dual-gene edited pig kidneys can survive up to 499 days after transplantation into non-human primates, suggesting that while knocking out xenoantigen genes and transferring human complement regulatory proteins such as CD55 into the pig genome, it can effectively alleviate immune rejection reactions caused by complement activation and prolong the survival of xenotransplanted kidneys.

[0026] Beneficial effects of the present invention: The present invention designs two different sgRNA targeting vectors for the GGTA1 gene and connects them to the backbone vectors respectively, thereby overcoming the problem of low production efficiency of cloned pigs after simultaneous modification of multiple genes in the CRISPR / Cas9 gene editing technology.

[0027] The present invention discloses a method for constructing a four-gene edited xenotransplantation donor pig. The method can efficiently obtain the four-gene edited xenotransplantation donor pig of GTKO / hCD39 / hCD55 / hTBM by modifying different gene combinations, and can also provide technical support for solving the common problems such as immunological incompatibility and physiological functional disorders faced by pig-human xenotransplantation, lay a foundation for the research on developing xenotransplantation donor pigs suitable for different cells, tissues and organs, and is of great significance for promoting the clinical transformation and application of pig-human xenotransplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the technical roadmap of the present invention.

[0029] Figure 2 Schematic diagram of the construction of the GTKO / hCD39 / hCD55 / hTBM four-gene editing vector.

[0030] Figure 2 A in the middle is a schematic diagram of GGTA1 gene knockout and hCD39 / hCD55 / hTBM gene overexpression.

[0031] Figure 2 Aa in the figure is a schematic diagram of the construction of the GGTA1 gene knockout vector.

[0032] Figure 2 Ab in the figure is a schematic diagram of the construction of the hCD55 gene overexpression vector.

[0033] Figure 2 Ac in the figure is a schematic diagram of the construction of hCD39 / hTBM gene overexpression vector.

[0034] Figure 2 Middle B is the Sanger sequencing result of GGTA1 knockout in monoclonal porcine fetal fibroblasts.

[0035] Figure 2 Middle C is the PCR detection result of the monoclonal porcine fetal fibroblast hCD39 / hTBM gene expression.

[0036] Figure 3 This is the identification result of cloned pig fetus with four genes edited: GTKO / hCD39 / hCD55 / hTBM.

[0037] Figure 3 A in the middle is a pig fetus cloned by editing four genes: GTKO / hCD39 / hCD55 / hTBM.

[0038] Figure 3 Middle B is the PCR identification result of overexpression of hCD39, hCD55 and hTBM genes in cloned pig fetus.

[0039] Figure 3 Middle C is the PCR amplification result of GGTA1 gene.

[0040] Figure 3 D in the middle is the result of T7ENI restriction enzyme digestion identification of the PCR product of GGTA1 gene.

[0041] Figure 3 Middle E is the Sanger sequencing result of the PCR product of GGTA1 gene.

[0042] Figure 3 F in the middle is the result of immunoblotting identification of hCD39, hCD55, and hTBM gene overexpression.

[0043] Figure 4 This is the identification result of mRNA and protein expression level of four-gene edited cloned pigs GTKO / hCD39 / hCD55 / hTBM.

[0044] Figure 4 A in the middle is a cloned pig with four genes edited: GTKO / hCD39 / hCD55 / hTBM.

[0045] Figure 4 Figure B shows the identification results of the mRNA expression levels of the four gene-edited cloned pigs GGTA1, hCD39, hCD55, and hTBM genes.

[0046] Figure 4Center C shows the results of flow cytometry identification of the gene expression of four gene-edited cloned pigs: GGTA1, hCD39, hCD55, and hTBM.

[0047] Figure 4 D in the middle shows the immunofluorescence identification results of the gene expression of four gene-edited cloned pigs: GGTA1, hCD39, hCD55, and hTBM.

[0048] Figure 4 Middle E shows the identification results of the protein expression levels of the four gene-edited cloned pigs GGTA1, hCD39, hCD55, and hTBM genes. DETAILED DESCRIPTION

[0049] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below to facilitate understanding by technicians.

[0050] Example 1: Construction of GTKO / hCD39 / hCD55 / hTBM four-gene editing targeting vector (1) Construction of GTKO gene editing expression vector based on CRISPR / Cas9 gene editing system The nucleotide sequence of the porcine GGTA1 (Gene ID: 396733) gene was found in the NCBI database. The online software (http: / / crispor.tefor.net / ) was used to design and screen the targeting sgRNA sites for the 8th exon of the GGTA1 gene on porcine chromosome 1, and the sgRNA nucleotide sequence was connected to the backbone vector to obtain the GGTA1 gene targeting sgRNA expression vectors GGTA1-sgRNA1 (nucleotide sequence: 5′-GCTACAGGCCTGGTGGTACA-3′) and GGTA1-sgRNA2 (nucleotide sequence: 5′-GATGCGCATGAAGACCATCG-3′) ( Figure 2 (Aa in Chinese).

[0051] (2) Based on the piggyBac system, construct hCD39, hCD55, and hTBM humanized gene transfer expression vectors Based on the piggyBac system, the nucleotide sequences of hCD39 (SEQ ID NO: 3), hCD55 (SEQ ID NO: 4), and hTBM (SEQID NO: 5) genes were connected to the piggyBac backbone vector to obtain hCD55 ( Figure 2 Ab) and hCD39, hTBM ( Figure 2The nucleotide sequence used to transfer the hCD55 gene recombinant plasmid is shown in SEQ ID NO:6; the nucleotide sequence used to transfer the hCD39 and hTBM gene recombinant plasmid is shown in SEQ ID NO:7.

[0052] Example 2: Construction of GTKO / hCD39 / hCD55 / hTBM four-gene edited cloned pigs Targeting exon 8 of the GGTA1 gene (Gene ID: 396733) on chromosome 1 of pigs, the online software (http: / / crispor.tefor.net / ) was used to design and screen the targeting sgRNA sites, and the nucleotide sequences of sgRNA1 and sgRNA2 were connected to the backbone vector (PX458-sgRNA-EGFP) to obtain the GGTA1 gene knockout recombinant plasmid vectors GGTA1-sgRNA1 (SEQ ID NO: 1) and GGTA1-sgRNA2 (SEQ ID NO: 2), respectively. Based on the piggyBac system, the nucleotide sequences of hCD39 (SEQ ID NO: 3), hCD55 (SEQ ID NO: 4), and hTBM (SEQ ID NO: 5) genes were connected to the piggyBac transposon to obtain the recombinant hCD55 gene transfer expression vector (SEQ ID NO: 6) and hCD39, hTBM gene transfer expression vector (SEQ ID NO: 7), respectively. The GGTA1 gene knockout targeting vector and the hCD39, hCD55, and hTBM humanized gene transfer expression vector were co-transfected into the wild-type porcine fetal fibroblast cell line under the action of transposase. After 48 hours of puromycin maintenance and screening, a total of 107 single cell clones were obtained after extreme dilution culture. PCR identification showed that the hCD39, hCD55, and hTBM genes were successfully transferred ( Figure 2 C), the transfer success rate was 100%, and Sanger sequencing results showed that the GGTA1 gene in C17#, C21#, C26#, C29#, C57#, C63#, and C78# single cell clones had eight gene editing types: +1bp, +2bp, +3bp, -3bp, -4bp, -26bp, -27bp, and -420bp ( Figure 2 In the figure (B), positive single cell clones (C21# and C78#) with double knockout of GGTA1 gene and successful transfer of hCD39, hCD55 and hTBM were selected as donor cells for somatic cell nuclear transplantation to construct cloned embryos, which were then transplanted into estrus surrogate sows. Four-gene edited cloned pigs were obtained by natural delivery after 114 days of gestation ( Figure 4As a priority, the fetus can be removed by caesarean section at 33 days of gestation from the surrogate sow, and the fetal fibroblast cell line can be isolated and the genotype identified. This can be further used as a donor cell for somatic cell nuclear transplantation to produce four-gene edited cloned pigs, thereby improving the success rate and production efficiency of the construction of four-gene edited cloned pigs.

[0053] Example 3: Genotyping and phenotypic analysis of four-gene edited cloned pigs The fibroblast cell line of 33-day-old cloned pig fetus (A in Figure 3) was isolated, and the genomic DNA of the cloned pig fetus was extracted. The genotype was identified by PCR, T7ENI, Sanger sequencing and other technologies. The results showed that all 7 fetuses were cloned pig fetuses with four genes edited: GGTA1, hCD39, hCD55, and hTBM ( Figure 3 The editing success rate of cloned fetuses was 100%. The related protein expression levels of the four gene-edited cloned pig fetal fibroblast cell lines were further analyzed by Western blotting technology. The results showed that the GGTA1 gene was successfully knocked out in the 2# cloned pig fetus, and the hCD39, hCD55, and hTBM genes were successfully expressed ( Figure 3 F). Further, secondary somatic cell nuclear transplantation and embryo transplantation were performed using 2# cloned pig fetal fibroblasts as donor cells to construct a four-gene edited cloned pig ( Figure 4 A), q-RCR technology, flow cytometry, tissue immunofluorescence technology, and Western blotting molecular biology technology were further used to detect the mRNA and protein expression levels of GGTA1, hCD39, hCD55, and hTBM genes in the heart, liver, kidney, lung tissues, and fibroblast cell lines of the four gene-edited cloned pigs. The results showed that the GGTA1 antigen was completely knocked out in the four gene-edited cloned pigs, and the hCD39, hCD55, and hTBM humanized genes were successfully expressed ( Figure 4 BE in the middle), indicating that the GTKO / hCD39 / hCD55 / hTBM four-gene edited xenotransplantation donor pig was successfully constructed.

[0054] In Example 3, the four-gene-edited xenotransplantation donor pig constructed for the common problems in xenotransplantation can accelerate the research and development of xenotransplantation donor pigs and overcome the immune rejection reaction in the process of xenotransplantation. At the same time, based on the four-gene-edited xenotransplantation donor pig, different gene editing strategies can be adopted to modify the target gene in response to the scientific problems faced by xenotransplantation of different cells, tissues, and organs, which has important scientific application value for the development of truly effective xenotransplantation donor pigs using multi-gene combination modification methods.

[0055] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. GGTA1 gene targeting sgRNA expression vector, characterized by: The GGTA1 gene targeting sgRNA expression vectors GGTA1-sgRNA1 and GGTA1-sgRNA2 were constructed using CRISPR / Cas9 gene editing technology. The action site of sgRNA is located in the 8th exon of the GGTA1 gene.

2. The GGTA1 gene targeting sgRNA expression vector according to claim 1, characterized in that: The nucleotide sequence of GGTA1-sgRNA1 is: 5′-GCTACAGGCCTGGTGGTACA-3′; the nucleotide sequence of GGTA1-sgRNA2 is: 5′-GATGCGCATGAAGACCATCG-3′.

3. The GGTA1 gene targeting sgRNA expression vector according to claim 1 or 2, characterized in that: The backbone vector to which GGTA1-sgRNA1 and GGTA1-sgRNA2 are connected is PX458-sgRNA-EGFP, and its gene number is: Addgene no: 112220.

4. A recombinant plasmid for knocking out the GGTA1 gene in the pig genome, characterized in that: Recombinant plasmids include: PX458-GGTA1-sgRNA1 and PX458-GGTA1-sgRNA2; the nucleotide sequence of PX458-GGTA1-sgRNA1 is shown in SEQ ID NO: 1; the nucleotide sequence of PX458-GGTA1-sgRNA2 is shown in SEQ ID NO:

2.

5. hCD39, hCD55, hTBM humanized gene expression vector, characterized by: It includes a recombinant plasmid for transferring hCD55 gene and a recombinant plasmid for transferring hCD39 and hTBM genes together.

6. The humanized gene expression vector of hCD39, hCD55 and hTBM according to claim 5, characterized in that: The nucleotide sequence of the recombinant plasmid used to transfer the hCD55 gene is shown in SEQ ID NO:

6.

7. The humanized gene expression vector of hCD39, hCD55, or hTBM according to claim 5 or 6, characterized in that: The nucleotide sequence of the recombinant plasmid used for co-transferring hCD39 and hTBM genes is shown in SEQ ID NO:

7.

8. Construction of a GTKO / hCD39 / hCD55 / hTBM four-gene edited porcine fetal fibroblast cell line, characterized by: The GGTA1 gene targeting sgRNA expression vector described in any one of claims 1 to 3, or the recombinant plasmid for knocking out the GGTA1 gene in the pig genome described in claim 4, and / or the humanized gene expression vector described in any one of claims 5 to 7 are co-transfected into a pig fetal fibroblast cell line under the action of a transposase to obtain a four-gene edited positive cell monoclone, namely, the GTKO / hCD39 / hCD55 / hTBM four-gene edited pig fibroblast cell line.

9. A method for constructing a four-gene edited xenotransplantation donor pig, characterized in that: The specific steps are: 1) Construction of a recombinant plasmid for knocking out the GGTA1 gene in the pig genome Targeting sgRNA1 and sgRNA2 were designed for exon 8 of the porcine GGTA1 gene and connected to the backbone vectors, respectively, to obtain a recombinant plasmid for knocking out the GGTA1 gene in the porcine genome, including: PX458-GGTA1-sgRNA1 and PX458-GGTA1-sgRNA2; the nucleotide sequence of PX458-GGTA1-sgRNA1 is shown in SEQ ID NO: 1; the nucleotide sequence of PX458-GGTA1-sgRNA2 is shown in SEQ ID NO: 2; 2) Construction of humanized gene expression vectors for hCD39, hCD55, and hTBM based on the piggyBac system Based on the piggyBac system, the humanized gene expression vectors of hCD39, hCD55, and hTBM were constructed using homologous recombination technology; 3) Construction of GTKO / hCD39 / hCD55 / hTBM four-gene edited porcine fibroblast cell line The recombinant plasmid for knocking out the GGTA1 gene in the pig genome, the hCD55 humanized gene expression vector, and the hCD39 / hTBM humanized gene expression vector were co-transfected into the wild-type porcine fetal fibroblast cell line, and the GTKO / hCD39 / hCD55 / hTBM four-gene edited porcine fibroblast cell line was screened and obtained through single-cell cloning genotype identification; 4) Somatic cell nuclear transfer and embryo transfer Using somatic cell nuclear transfer technology, the GTKO / hCD39 / hCD55 / hTBM four-gene edited porcine fetal fibroblast cell line obtained by transfection screening is subjected to somatic cell nuclear transfer, and the fetus is removed during pregnancy to identify the genotype and isolate and culture the cloned porcine fetal fibroblast cell line, or the four-gene edited cloned pig is obtained when the fetus develops to birth; 5) Genotyping and phenotypic analysis of cloned pigs The genomic DNA of cloned pig tissues was extracted, and its genotype was identified by PCR, T7ENI, and Sanger sequencing. The expression levels of gene mRNA and protein in tissues, organs, and cells were further detected by q-PCR, flow cytometry, tissue immunofluorescence, and Western blotting. 6) Using continuous cloning technology, the four-gene edited porcine fetal fibroblast cell line or porcine fibroblast cell line obtained in step 4) above is subjected to somatic cell nuclear transplantation, and the cloned embryos are transplanted into surrogate sows to mass-produce GTKO / hCD39 / hCD55 / hTBM four-gene edited xenotransplantation donor pigs that have been protein-identified.

10. The GGTA1 gene targeting sgRNA expression vector according to any one of claims 1 to 3, the recombinant plasmid for knocking out the GGTA1 gene in the pig genome according to claim 4, the humanized gene expression vector according to any one of claims 5 to 7, the four-gene edited pig or pig fetal fibroblast cell line according to claim 8, and / or the method for constructing a four-gene edited xenotransplantation donor pig according to claim 9, in pig-non-human primate xenotransplantation.

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