Construction method of eight-gene edited xenogenous organ transplantation donor pig

Abstract

The invention relates to a construction method of an eight-gene edited xenogenic organ transplantation donor pig, and belongs to the technical field of animal biology. In a wild type pig fetal fibroblast cell line, GGTA1, beta4GalNT2 and CMAH genes are knocked out by utilizing a CRISPR / Cas9 gene editing technology, hCD39, hCD46, hCD55, hCD59 and hTBM humanized genes are transferred, a GTKO / beta4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene editing cloned pig is constructed in combination with a somatic cell cloning technology, and an eight-gene editing xenograft donor pig is further obtained through genotype, mRNA and protein expression identification and functional analysis. According to the method, the technical problems of high production difficulty, low efficiency and low survival rate of the multi-gene editing xenogenic organ transplantation donor pig are solved, and the common problems of immunological rejection and complement disorder in the xenogenic organ transplantation process are solved to the greatest extent; a foundation is laid for developing donor pigs suitable for xenotransplantation of different tissues and organs in a more targeted manner, and the method has important value for promoting clinical transformation of xenotransplantation of the organs.

Description

Technical Field

[0001] The present invention belongs to the field of animal biotechnology, and specifically relates to a method for constructing an eight-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. About 200,000 people around the world wait for organ transplantation each year, 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 world's public health field. Xenotransplantation has been recognized as an effective way to solve the global gap in human organ transplantation supply and demand. 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. The patient survived for 2 months after the transplant. In August 2023, the University of Alabama at Birmingham transplanted a 10-gene-edited pig kidney into the human body and it can function 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. After the transplant, the patient could breathe independently and survived successfully for 6 weeks. Pig-human xenotransplantation is hailed as the "next major biomedical revolution", marking the most critical step in the clinical application of pig-human xenotransplantation.

[0003] Xenotransplantation still needs to overcome rejection, control inflammation, regulate coagulation disorders and other problems. In order to overcome these obstacles, the genetic manipulation of xenotransplantation mainly includes two purposes: (I) inactivation of pig-derived xenoantigens; (II) transgenic expression of human protective proteins, including coagulation regulatory factors, complement regulatory factors, cellular immune response factors, anti-apoptosis and anti-inflammatory factors. In recent years, with the advancement of gene editing technology, various gene-edited donor pigs have been reported. The results of subclinical studies on pig-human xenotransplantation have also effectively proved that adding some other xenoantigen gene knockouts and humanized gene expression on the basis of GGTA1 gene knockout can help reduce the immune rejection caused by xenotransplantation. For these major xenoantigen genes and a large number of humanized gene expression modifications, the existing gene editing technology applications generally have common problems such as difficulty in editing multiple genomes at the same time, low efficiency, low survival rate of multi-gene edited cloned pigs, and uneven expression of target genes. Summary of the invention

[0004] In view of the above problems, the present invention provides a method for constructing an eight-gene edited xenotransplantation donor pig. The method aims at the common problem of immune rejection reaction in the process of xenotransplantation, and utilizes CRISPR / Cas9 gene editing technology and piggyBac transposon technology, combined with somatic cell cloning technology to construct an eight-gene edited xenotransplantation donor pig of GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM, which is of great significance for the preclinical research of pig-non-human primate xenotransplantation and the clinical application of pig-human xenotransplantation.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions: In a first aspect, the present invention provides sgRNA targeting vectors of GGTA1, β4GalNT2, and CMAH genes, which utilize CRISPR / Cas9 gene editing technology to construct sgRNA targeting vectors of GGTA1, β4GalNT2, and CMAH genes, including: GGTA1-sgRNA1, GGTA1-sgRNA2, β4GalNT2-sgRNA1, β4GalNT2-sgRNA2, β4GalNT2-sgRNA3, CMAH-sgRNA1, and CMAH-sgRNA2.

[0006] Furthermore, the above-mentioned sgRNA action site is located in the 3rd exon of the porcine GGTA1 gene, the 2nd exon of the β4GalNT2 gene, and the 4th exon of the CMAH gene.

[0007] Furthermore, the nucleotide sequence of the above-mentioned GGTA1-sgRNA1 is shown in SEQ ID NO: 1; the nucleotide sequence of GGTA1-sgRNA2 is shown in SEQ ID NO: 2; the nucleotide sequence of β4GalNT2-sgRNA1 is shown in SEQ ID NO: 3; the nucleotide sequence of β4GalNT2-sgRNA2 is shown in SEQ ID NO: 4; the nucleotide sequence of β4GalNT2-sgRNA3 is shown in SEQ ID NO: 5; the nucleotide sequence of CMAH-sgRNA1 is shown in SEQ ID NO: 6; the nucleotide sequence of CMAH-sgRNA2 is shown in SEQ ID NO: 7.

[0008] Furthermore, the backbone vector connected to the above-mentioned GGTA1, β4GalNT2, and CMAH gene sgRNA targeting vectors is PX458-sgRNA-EGFP (Addgene no: 112220).

[0009] The second aspect of the present invention provides a recombinant plasmid for knocking out the GGTA1, β4GalNT2 and CMAH genes encoded in the pig genome, and the nucleotide sequence thereof is shown in SEQ ID NO:8.

[0010] The third aspect of the present invention provides an expression vector for transferring the humanized genes of hCD39, hCD46, hCD55, hCD59 and hTBM.

[0011] Furthermore, the nucleotide sequence of hCD39 is shown in SEQ ID NO:9; the nucleotide sequence of hCD46 is shown in SEQ ID NO:10; the nucleotide sequence of hCD55 is shown in SEQ ID NO:11; the nucleotide sequence of hCD59 is shown in SEQ ID NO:12; and the nucleotide sequence of hTBM is shown in SEQ ID NO:13.

[0012] Furthermore, the backbone vector to which the humanized genes of hCD39, hCD46, hCD55, hCD59 and hTBM are transferred into the expression vector is the PiggyBac transposon.

[0013] Furthermore, the nucleotide sequence of the hCD39, hCD46, hCD55, hCD59, and hTBM humanized genes constructed based on the PiggyBac transposon system and transferred into the expression vector is shown in SEQ ID NO:14.

[0014] The fourth aspect of the present invention provides an eight-gene edited porcine fetal fibroblast cell line GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM, wherein the sgRNA targeting vectors of GGTA1, β4GalNT2, and CMAH genes and the recombinant plasmid for knocking out the GGTA1, β4GalNT2, and CMAH genes in the porcine genome are co-transfected into wild-type porcine fetal fibroblasts under the action of transposase, and the eight-gene edited positive monoclonal cell line obtained by screening is the eight-gene edited porcine fetal fibroblast cell line GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM.

[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 an eight-gene edited xenotransplantation donor pig, the specific steps of which are: (1) Construction of GTKO / β4GalNT2KO / CMAHKO gene targeting vector based on CRISPR / Cas9 gene editing system Aiming at the 3rd exon of GGTA1 gene, the 2nd exon of β4GalNT2 gene and the 4th exon of CMAH gene encoded in the pig genome, sgRNA targeting sequences were designed and connected to the backbone vector to obtain GTKO, β4GalNT2KO and CMAHKO gene sgRNA targeting vectors; wherein, 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; the nucleotide sequence of β4GalNT2-sgRNA1 is shown in SEQ ID NO:3; the nucleotide sequence of β4GalNT2-sgRNA2 is shown in SEQ ID NO:4; the nucleotide sequence of β4GalNT2-sgRNA3 is shown in SEQ ID NO:5; the nucleotide sequence of CMAH-sgRNA1 is shown in SEQ ID NO:6; the nucleotide sequence of CMAH-sgRNA2 is shown in SEQ ID NO:7. NO:7; the nucleotide sequence of the targeting sgRNA recombinant plasmid for knocking out GGTA1, β4GalNT2, and CMAH genes is shown in SEQ ID NO:8.

[0017] (2) Construction of humanized gene transfer expression vectors for hCD39 / hCD46 / hCD55 / hCD59 / hTBM based on the piggyBac transposon system The humanized genes of hCD39, hCD46, hCD55, hCD59 and hTBM constructed based on the piggyBac transposon system were transferred into the expression vector, and the nucleotide sequence of hCD39 is shown in SEQ ID NO:9; the nucleotide sequence of hCD46 is shown in SEQID NO:10; the nucleotide sequence of hCD55 is shown in SEQ ID NO:11; the nucleotide sequence of hCD59 is shown in SEQID NO:12; the nucleotide sequence of hTBM is shown in SEQ ID NO:13; the nucleotide sequence of the humanized genes of hCD39, hCD46, hCD55, hCD59 and hTBM transferred into the expression vector is shown in SEQID NO:14.

[0018] (3) Construction of eight-gene edited porcine fetal fibroblast cell line: GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM The GGTA1, β4GalNT2, CMAH gene sgRNA targeting vectors and the hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized genes are transferred into the expression vector and co-transfected into the wild-type porcine fetal fibroblast cell line under the action of transposase. The transfection method includes liposome transfection or electrofection, preferably electrofection. After monoclonal cell genotype identification, the eight-gene edited porcine fetal fibroblast cell line GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM is screened and obtained.

[0019] (4) Somatic cell nuclear transfer and embryo transfer Somatic cell nuclear transplantation is performed using GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited positive monoclonal fibroblasts or cloned fetal fibroblasts or cloned porcine fibroblasts, preferably cloned porcine fetal fibroblasts, to construct GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned embryos, which are further transplanted into estrus surrogate sows for development, and GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned pigs are obtained by natural delivery after 114 days of gestation, or the fetus is removed during pregnancy for genotype identification and establishment of a fetal fibroblast cell line.

[0020] (5) Genotyping and phenotypic analysis of cloned pigs The genomic DNA of cloned piglets was extracted, and the genotype of cloned pigs was identified by PCR, T7ENI, and Sanger sequencing techniques, and eight-gene edited cloned pigs (GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM) were obtained. The mRNA and protein expression levels of different tissues and organs of the eight-gene edited cloned pigs were further detected by Western blotting, immunofluorescence staining, flow cytometry, HE staining, and cross-matching molecular biology techniques.

[0021] 6) Using continuous cloning technology, the eight-gene edited porcine fetal fibroblast cell line obtained in step 3) or the eight-gene edited porcine fibroblast cell line obtained in step 5) is subjected to somatic cell nuclear transplantation, and the cloned embryos are transplanted into surrogate sows. After protein identification, GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited xenotransplantation donor pigs are mass-produced.

[0022] The above-mentioned GGTA1, β4GalNT2, CMAH gene sgRNA targeting vectors, recombinant plasmids for knocking out GGTA1, β4GalNT2, CMAH genes in the pig genome, hCD39, hCD46, hCD55, hCD59, hTBM humanized gene transfer expression vectors, GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited pig fetal fibroblast cell lines, and / or, the application of the eight-gene edited xenotransplantation donor pig construction method in the field of xenotransplantation biomedical research.

[0023] In order to overcome the rejection, inflammation, coagulation disorders and other obstacles in xenotransplantation, genetic manipulation of xenotransplantation donor pigs mainly includes two purposes: (I) inactivation of pig-derived xenoantigens; and (II) transgenic expression of human protective proteins, including coagulation regulatory factors, complement regulatory factors, cellular immune response factors, anti-apoptosis and anti-inflammatory factors, etc.

[0024] Inactivation of GGTA1 (producing α-Gal antigen), CMAH (producing Neu5Gc antigen) and β4GalNT2 (producing Sda antigen) in pigs can effectively reduce the occurrence of hyperacute and acute rejection reactions mediated by antigen-antibody in xenotransplantation of kidneys. Knocking out CMAH and β4GalNT2 genes on the basis of knocking out GGTA1 gene will more effectively alleviate immune rejection after xenotransplantation. Compared with cells with single GGTA1 gene and GGTA1 / CMAH gene deficiency, GGTA1 / CMAH / β4GALNT2 triple-gene knockout pig cells have significantly reduced binding to xenogeneic IgM and IgG in vitro, indicating that GGTA1 / CMAH / β4GalNT2 triple-gene knockout pigs have lower immunogenicity and can better ensure the long-term survival of xenotransplants.

[0025] The expression of human complement regulatory proteins (CD46, CD55, CD59) and thrombomodulin (CD39, TBM) in xenotransplant donor pigs can correct the disorders of complement and coagulation caused by species incompatibility. Compared with GTKO single-gene edited pigs, the survival time of GTKO / hCD55 dual-gene edited pig kidneys transplanted into non-human primates can reach 499 days, indicating that knocking out the xenoantigen gene and transferring human complement regulatory proteins such as CD46, CD55, CD59 into the pig can effectively alleviate the rejection reaction caused by complement activation and prolong the survival of xenotransplanted kidneys.

[0026] The results effectively proved that adding other xenoantigen gene knockouts and humanized gene expression on the basis of GGTA1 knockout can help reduce the immune rejection reaction caused by xenotransplantation. Therefore, the GTKO / CMAHKO / β4GalNT2KO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited xenotransplantation donor pig constructed by the present invention can help solve the antigen-antibody mediated immune rejection reaction faced by pig-human xenotransplantation, and is of great significance for the preclinical research of pig-non-human primate xenotransplantation and the clinical trial of pig-human xenotransplantation.

[0027] Beneficial effects of the present invention: The present invention designs multiple different sgRNA nucleotide series for gene knockout targeting vectors, and constructs expression vectors for simultaneous editing of multiple genes. While improving the success rate of gene targeting, it overcomes the common technical difficulties of CRISPR / Cas9 technology, such as low efficiency of multi-gene editing, low birth efficiency of cloned pigs after multi-gene modification, and difficulty in survival.

[0028] The present invention discloses a method for constructing an eight-gene edited xenotransplantation donor pig. By modifying different gene combinations, three carbohydrate antigen genes in the pig genome are inactivated and humanized genes are transferred into the pig genome for overexpression. This solves the common problems such as hyperacute immune rejection reaction and antigen-antibody mediated immune rejection reaction in the process of xenotransplantation to a certain extent, lays a foundation for further and more targeted development of donor pigs suitable for xenotransplantation of tissues or organs such as heart, kidney, liver, and skin, and has important prospects for conducting preclinical research on pig-non-human primate xenotransplantation and clinical application of pig-human xenotransplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of eight-gene edited cloned pigs and vector construction: GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM; Figure 1 A in the middle is a schematic diagram of the construction of eight-gene edited cloned pigs: GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM; Figure 1 Middle B is a schematic diagram of GTKO / β4GalNT2KO / CMAHKO gene knockout targeting; Figure 1 Center C is a schematic diagram of the construction of the CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM gene editing vector.

[0030] Figure 2 These are the results of PCR identification of gene editing in the monoclonal porcine fibroblast cell line hCD39 / hCD46 / hCD55 / hCD59 / hTBM.

[0031] Figure 3 The results of gene editing PCR and Sanger sequencing identification of the monoclonal porcine fibroblast cell line GTKO / β4GalNT2KO / CMAHKO; Figure 3 A in the middle is the PCR identification result of GGTA1, β4GalNT2, and CMAH genes in the monoclonal porcine fibroblast cell line; Figure 3 Figure B shows the results of Sanger sequencing of PCR products of GGTA1, β4GalNT2, and CMAH genes in the monoclonal porcine fibroblast cell line.

[0032] Figure 4 The genotype identification results of the eight-gene edited cloned pig fetus: GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM; Figure 4 A in the middle is a cloned pig fetus with eight genes edited: GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM; Figure 4 Middle B is the PCR identification result of GGTA1, β4GalNT2, and CMAH genes in the cloned pig fetus genome; Figure 4 Center C is the Sanger sequencing identification result of the PCR products of GGTA1, β4GalNT2, and CMAH genes in the cloned pig fetus genome.

[0033] Figure 5 The results of identification of mRNA and protein expression levels of eight gene-edited cloned pigs: GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM.

[0034] Figure 5 A in the middle is a cloned pig with eight genes edited: GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM; Figure 5 Middle B is the expression level of hCD46 gene mRNA in cloned pig heart, liver, kidney and lung; Figure 5 Middle C is the expression level of hCD55 gene mRNA in cloned pig heart, liver, kidney and lung; Figure 5 D in the middle is the expression level of hCD59 gene mRNA in cloned pig heart, liver, kidney and lung; Figure 5 Middle E is the expression level of hTBM gene mRNA in cloned pig heart, liver, kidney, and lung; Figure 5 Middle F is the expression level of hCD39 gene mRNA in cloned pig heart, liver, kidney, and lung; Figure 5 Middle G is the result of immunofluorescence detection of GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM gene expression in cloned pig tissues; Figure 5 Middle H represents the binding ability of eight gene-edited cloned pig peripheral blood mononuclear cells to monkey IgG; Figure 5 Middle I is the binding ability of eight gene-edited cloned pig peripheral blood mononuclear cells to monkey IgM; Figure 5 Middle J is a monkey complement-dependent cytotoxicity experiment analyzing the survival level of eight gene-edited cloned pig cells. DETAILED DESCRIPTION

[0035] 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.

[0036] Example 1: Construction of eight gene editing targeting vectors (1) Construction of GTKO / β4GalNT2KO / CMAHKO gene targeting vector based on CRISPR / Cas9 gene editing technology The nucleotide sequences of porcine GGTA1 (Gene ID: 396733), β4GalNT2 (Gene ID: 100621328), and CMAH (Gene ID: 396918) genes were obtained from the NCBI database. Targeting sgRNA sites were designed and screened for exon 3 of porcine GGTA1 gene, exon 2 of β4GalNT2 gene, and exon 4 of CMAH gene using online software (http: / / crispor.tefor.net / ), and sgRNA was ligated to the backbone vector PX458-sgRNA-EGFP (Addgene no: 112220), and GGTA1-sgRNA1 (SEQ ID NO: 1), GGTA1-sgRNA2 (SEQ ID NO: 2), β4GalNT2-sgRNA1 (SEQ ID NO: 3), β4GalNT2-sgRNA2 (SEQ ID NO: 4), β4GalNT2-sgRNA3 (SEQ ID NO: 5), and sgRNA4 (SEQ ID NO: 6) were obtained, respectively. NO:5), CMAH-sgRNA1 (SEQ ID NO:6), CMAH-sgRNA2 (SEQ ID NO:7) targeting vector ( Figure 1 Middle B).

[0037] (2) Construction of hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized gene transfer expression vector based on piggyBac system Based on the piggyBac system, the nucleotide sequences of hCD39 (SEQ ID NO: 9), hCD46 (SEQ ID NO: 10), hCD55 (SEQ ID NO: 11), hCD59 (SEQ ID NO: 12), and hTBM (SEQ ID NO: 13) genes were connected to the piggyBac transposon vector to obtain the hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized gene transfer expression vector (SEQ ID NO: 14) ( Figure 1 Middle C).

[0038] Example 2: Construction of eight-gene edited cloned pigs Targeting exon 3 of porcine GGTA1 gene (Gene ID: 396733), exon 2 of β4GalNT2 gene (Gene ID: 100621328), and exon 4 of CMAH gene (Gene ID: 396918), online software (http: / / crispor.tefor.net / ) was used to design and screen targeting sgRNA sites, and the sgRNA sequences were connected to the backbone vector (PX458-sgRNA-EGFP) to obtain GGTA1-sgRNA1 (SEQ ID NO: 1), GGTA1-sgRNA2 (SEQ ID NO: 2), β4GalNT2-sgRNA1 (SEQ ID NO: 3), β4GalNT2-sgRNA2 (SEQ ID NO: 4), β4GalNT2-sgRNA3 (SEQ ID NO: 5), CMAH-sgRNA1 (SEQ ID NO: 6), and CMAH–sgRNA2 (SEQ ID NO: 7) targeting vectors ( Figure 1 Based on the piggyBac system, the nucleotide sequences of hCD39 (SEQ ID NO: 9), hCD46 (SEQ ID NO: 10), hCD55 (SEQ ID NO: 11), hCD59 (SEQ ID NO: 12), and hTBM (SEQ ID NO: 13) genes were connected to the piggyBac transposon to obtain the hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized gene transfer expression vector (SEQ ID NO: 10) ( Figure 1 C). The sgRNA targeting vectors of GGTA1, β4GalNT2, and CMAH genes and the humanized genes of hCD39, hCD46, hCD55, hCD59, and hTBM were transferred into the expression vector and 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 25 single cell clones were obtained after extreme dilution culture. After genotype identification, the eight-gene edited porcine fetal fibroblast cell line of GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM was successfully obtained. The C12# positive monoclonal cell line was used as the donor cell for somatic cell nuclear transplantation to construct cloned embryos, which were then transplanted into estrus surrogate sows. After 114 days of gestation, natural delivery was achieved to obtain eight-gene edited cloned pigs ( Figure 5 Middle A). When the surrogate sow was 33 days pregnant, a live fetus was obtained by caesarean section ( Figure 4Middle A), the fibroblast cell line was isolated and the gene editing of GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM was identified. The results showed that GGTA1, β4GalNT2, and CMAH were all biallelic knockouts ( Figure 4 In BC), the success rate of eight-gene editing in cloned pig fetuses was 100%, and somatic cell nuclear transplantation was further performed using them as donor cells to produce eight-gene edited cloned pigs.

[0039] Example 3: Genotyping and phenotypic analysis of eight-gene edited cloned pigs Using eight-gene edited fetal fibroblasts as donor cells, somatic cell nuclear transplantation was performed, and cloned embryos were transplanted into 17 surrogate sows. Six of them became pregnant. After 114 days of pregnancy, 28 cloned pigs were obtained through natural delivery, of which 24 survived ( Figure 5 The eight-gene-edited cloned pigs (A) had an individual survival rate of 85.71%. Human umbilical cord endothelial cells were used as positive controls to detect the expression of five humanized genes in the heart, kidney, liver and lung tissues of the eight-gene-edited piglets by q-PCR. Compared with the wild type, hCD46 ( Figure 5 Middle B), hCD55 ( Figure 5 Middle C), hCD59 ( Figure 5 Middle D), hTBM ( Figure 5 Middle E), hCD39 ( Figure 5 F) The gene mRNA showed different expression levels in different tissues such as heart, liver, kidney and lung. The knockout of three xenoantigens and the expression of five humanized proteins in the kidney tissue of the eight-gene-edited cloned pigs were verified by immunofluorescence staining. Compared with wild-type pigs, GGTA1, CMAH and β4GalNT2 were successfully inactivated in the kidney of the eight-gene-edited cloned pigs, and hTBM, hCD39, hCD46, hCD55 and hCD59 showed different expression patterns ( Figure 5 G), monkey serum was collected and cross-matched with PBMC cells of eight-gene-edited cloned pigs. Compared with wild-type pigs, the ability of peripheral blood mononuclear cells (PBMC) of eight-gene-edited cloned pigs to bind monkey IgG and IgM was significantly reduced ( Figure 5 HI), complement-dependent cytotoxicity experiments further confirmed that the eight-gene edited cloned pigs helped protect pig cells from complement attack, thereby increasing the survival rate of pig cells ( Figure 5 Middle J), ​​indicating that eight gene-edited xenotransplantation donor pigs were successfully constructed.

[0040] In the embodiment, the eight-gene-edited xenotransplantation donor pig constructed by the present invention can accelerate the research and development process of xenotransplantation donor pigs, and solve the common problems in the development of xenotransplantation donor pigs and xenotransplantation to the greatest extent. At the same time, based on the eight-gene-edited xenotransplantation donor pigs, different gene editing strategies can be adopted to modify related genes in response to the scientific problems faced by xenotransplantation of different cells, tissues, and organs, which has important application value for the development of effective xenotransplantation donor pigs using multi-gene combination modification.

[0041] 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, β4GalNT2, CMAH gene sgRNA targeting vector, characterized by: CRISPR / Cas9 gene editing technology was used to construct sgRNA targeting vectors for GGTA1, β4GalNT2, and CMAH genes. The sgRNA action sites were located in the 3rd exon of the porcine GGTA1 gene, the 2nd exon of the β4GalNT2 gene, and the 4th exon of the CMAH gene.

2. The GGTA1, β4GalNT2, CMAH gene sgRNA targeting vector according to claim 1, characterized in that: include: GGTA1-sgRNA1, GGTA1-sgRNA2, β4GalNT2-sgRNA1, β4GalNT2-sgRNA2, β4GalNT2-sgRNA3, CMAH-sgRNA1, CMAH-sgRNA2; 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; the nucleotide sequence of β4GalNT2-sgRNA1 is shown in SEQ ID NO: 3; the nucleotide sequence of β4GalNT2-sgRNA2 is shown in SEQ ID NO: 4; the nucleotide sequence of β4GalNT2-sgRNA3 is shown in SEQ ID NO: 5; the nucleotide sequence of CMAH-sgRNA1 is shown in SEQ ID NO: 6; the nucleotide sequence of CMAH-sgRNA2 is shown in SEQ ID NO:

7.

3. The GGTA1, β4GalNT2, CMAH gene sgRNA targeting vector according to claim 1 or 2, characterized in that: The backbone vector is PX458-sgRNA-EGFP, and its gene number is: Addgene no: 112220.

4. A recombinant plasmid for knocking out the GGTA1, β4GalNT2, and CMAH genes in the pig genome, characterized in that: The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO:

8.

5. hCD39, hCD46, hCD55, hCD59, and hTBM humanized genes are transferred into expression vectors, characterized in that: The humanized genes of hCD39, hCD46, hCD55, hCD59 and hTBM constructed based on the PiggyBac transposon system were transferred into the expression vector, and the nucleotide sequence thereof is shown in SEQ ID NO:

14.

6. GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line, characterized by: The GGTA1, β4GalNT2, CMAH gene sgRNA targeting vector described in any one of claims 1 to 3 or the recombinant plasmid for knocking out the GGTA1, β4GalNT2, and CMAH genes in the pig genome described in claim 4 are co-transfected into wild-type porcine fetal fibroblasts under the action of transposase, and the eight-gene edited positive monoclonal cell line obtained by screening is the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line.

7. A method for constructing eight-gene-edited xenotransplantation donor pigs, characterized in that: The specific steps are: (1) Construction of a recombinant plasmid for knocking out the GGTA1, β4GalNT2, and CMAH genes in the pig genome based on the CRISPR / Cas9 gene editing system Targeting the exon 3 of the GGTA1 gene, the exon 2 of the β4GalNT2 gene, and the exon 4 of the CMAH gene in the pig genome, sgRNA targeting vectors were designed and connected to the backbone vector to obtain GTKO, β4GalNT2KO, and CMAHKO gene sgRNA targeting vectors, including: GGTA1-sgRNA1, GGTA1-sgRNA2, β4GalNT2-sgRNA1, β4GalNT2-sgRNA2, β4GalNT2-sgRNA3, CMAH-sgRNA1, and CMAH-sgRNA2; (2) Construction of humanized gene transfer expression vectors for hCD39 / hCD46 / hCD55 / hCD59 / hTBM based on the piggyBac transposon system The humanized genes of hCD39, hCD46, hCD55, hCD59, and hTBM constructed based on the piggyBac transposon system were transferred into the expression vector, the nucleotide sequence of hCD39 is shown in SEQ ID NO:9; the nucleotide sequence of hCD46 is shown in SEQ ID NO:10; the nucleotide sequence of hCD55 is shown in SEQ ID NO:11; the nucleotide sequence of hCD59 is shown in SEQ ID NO:12; the nucleotide sequence of hTBM is shown in SEQ ID NO:13; (3) Construction of eight-gene edited porcine fetal fibroblast cell line: GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM The recombinant plasmid for knocking out the GGTA1, β4GalNT2, and CMAH genes in the pig genome and the hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized genes are transferred into an expression vector and co-transfected into a wild-type pig fetal fibroblast cell line under the action of a transposase, wherein the transfection method includes liposome transfection or electrofection, preferably electrofection, and the eight-gene edited pig fetal fibroblast cell line of GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM is screened and obtained through monoclonal cell genotype identification; (4) Somatic cell nuclear transfer and embryo transfer Using GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited positive monoclonal fibroblasts or cloned fetal fibroblasts or cloned porcine fibroblasts as donor cells, somatic cell nuclear transplantation is performed, with cloned fetal fibroblasts being preferred, to construct GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine cloned embryos, which are further transplanted into estrus surrogate sows for development, and GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned pigs are obtained by natural delivery, or the fetuses are removed during pregnancy for genotype identification and establishment of fibroblast cell lines; (5) Genotyping and phenotypic analysis of cloned pigs The genomic DNA of cloned piglets was extracted, and the genotype of cloned piglets was identified by PCR, T7ENI, and Sanger sequencing techniques. The mRNA and protein expression levels of different tissues and organs of eight-gene edited cloned pigs were detected by Western blotting, immunofluorescence staining, flow cytometry, HE staining, and cross-matching experimental molecular biology techniques, and the eight-gene edited cloned pigs GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM were obtained. (6) Using continuous cloning technology, the eight-gene edited porcine fetal fibroblast cell line obtained in step 3) or the eight-gene edited porcine fibroblast cell line obtained in step 5) is subjected to somatic cell nuclear transplantation, and the cloned embryos are transplanted into surrogate sows. After protein function identification, GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited xenotransplantation donor pigs are mass-produced.

8. The method for constructing a donor pig for eight-gene editing xenotransplantation according to claim 7, characterized in that: The nucleotide sequence of GGTA1-sgRNA1 in step 1) is shown in SEQ ID NO: 1; the nucleotide sequence of GGTA1-sgRNA2 is shown in SEQ ID NO: 2; the nucleotide sequence of β4GalNT2-sgRNA1 is shown in SEQ ID NO: 3; the nucleotide sequence of β4GalNT2-sgRNA2 is shown in SEQ ID NO: 4; the nucleotide sequence of β4GalNT2-sgRNA3 is shown in SEQ ID NO: 5; the nucleotide sequence of CMAH-sgRNA1 is shown in SEQ ID NO: 6; the nucleotide sequence of CMAH-sgRNA2 is shown in SEQ ID NO: 7; the nucleotide sequence of the recombinant plasmid used to knock out the GGTA1, β4GalNT2, and CMAH genes in the pig genome is shown in SEQ ID NO:

8.

9. A method for constructing an eight-gene edited xenotransplantation donor pig according to claim 7 or 8, characterized in that: The nucleotide sequence of the hCD39, hCD46, hCD55, hCD59, and hTBM humanized genes constructed based on the PiggyBac transposon system and transferred into the expression vector in step 2) is shown in SEQ ID NO:

14.

10. The sgRNA targeting vector of GGTA1, β4GalNT2, and CMAH genes according to any one of claims 1 to 3, the recombinant plasmid for knocking out GGTA1, β4GalNT2, and CMAH genes in the pig genome according to claim 4, the hCD39, hCD46, hCD55, hCD59, and hTBM humanized gene transfer expression vector according to claim 5, the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited pig fetal fibroblast cell line according to claim 6, and / or the method for constructing an eight-gene edited xenotransplantation donor pig according to any one of claims 7 to 9 in the field of biomedical research on xenotransplantation.

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