Multi-gene editing systems, cell lines and applications suitable for xenotransplantation

Abstract

This invention discloses a multi-gene editing system, cell line, and applications suitable for xenotransplantation. Through optimized multi-gene editing technology and system, this invention achieves efficient integration and stable expression of the deletion of three major antigens (GGTA1, CMAH, β4GALNT2) and seven human regulatory factors (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, EPCR), and obtained cell line No. 45. Genetically modified pigs prepared using this cell line No. 45 model can serve as xenotransplantation donor pigs. These donor pigs significantly reduce the recipient's immune rejection of xenotransplanted organs, greatly improving the survival time and physiological function persistence of xenotransplanted organs in the recipient body. This provides a standardized and systematic key technical pathway for the translation of xenotransplantation from basic experiments to clinical applications.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a multi-gene editing system, cell line, and applications suitable for xenotransplantation. Background Technology

[0002] With the development of medical technology, end-stage organ failure has become one of the most serious threats to human life and health. Currently, organ transplantation is the most effective treatment for the failure of vital organs such as the heart, kidneys, and liver. However, the source of human organs available for transplantation is extremely limited, with a severe shortage of donors. Every year, millions of patients die or suffer because they cannot receive timely transplants. Organ shortage has become a bottleneck problem that urgently needs to be solved by global healthcare systems.

[0003] Transplanting animal organs to humans (xenotransplantation) is considered a potential solution to the organ shortage. Among various animals, pigs are highly similar to humans in terms of body size, organ size, physiological function, and reproductive efficiency, and are considered one of the most promising animal sources for human organ donation. Therefore, using pigs as organ donors to conduct xenotransplantation research has become an international research hotspot.

[0004] However, direct transplantation of pig organs into humans triggers a strong immune rejection reaction. Research has found that one of the main reasons for this rejection is the presence of various specific carbohydrate antigens on the surface of pig cells that the human immune system cannot recognize or strongly reject. These antigens are rapidly recognized as "foreign substances" by the human immune system after transplantation, thereby activating an immune response and causing damage or even failure of the transplanted organ within a short period of time.

[0005] Therefore, there is an urgent need to research a new multi-gene editing system and cell line suitable for xenotransplantation to solve the above-mentioned problems in xenotransplantation. Summary of the Invention

[0006] The purpose of this invention is to provide a multi-gene editing system, cell line, and application suitable for xenotransplantation, in order to solve the above-mentioned problems.

[0007] According to a first aspect of the present invention, a multi-gene editing system suitable for xenotransplantation is provided, the multi-gene editing system comprising a recombinant DNA sequence capable of simultaneously knocking out three genes GGTA1, CMAH, and β4GALNT2 and site-specific integration of four genes hCD46, hCD55, hCD39, and hCD47; and a recombinant DNA sequence site-specific integration of three genes hCD59, hTBM, and EPCR. Therefore, by cloning the two recombinant DNA fragments into the expression vector, transfected cells can obtain cell lines with efficient integration and stable expression of the three major antigens (GGTA1, CMAH, β4GALNT2) and seven human regulatory factors (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, EPCR). Gene-edited pigs can be prepared from these cell lines and used as donors for xenotransplantation. This can reduce the recipient's immune rejection of xenotransplanted organs and greatly improve the survival time and physiological function of xenotransplanted organs in the recipient, providing research materials and new methods for xenotransplantation applications.

[0008] In some embodiments, the recombinant DNA fragment sequence is shown in SEQ ID NO:41 and SEQ ID NO:43.

[0009] In some embodiments, the multi-gene editing system includes an expression vector H11 pUC57 containing a DNA fragment as shown in SEQ ID NO:41 and an expression vector Rosa26 PUC57 containing a DNA fragment as shown in SEQ ID NO:43, wherein the sequence of the expression vector H11 pUC57 is shown in SEQ ID NO:42 and the sequence of the expression vector Rosa26 PUC57 is shown in SEQ ID NO:44.

[0010] According to a second aspect of the present invention, an application of a multi-gene editing system in the preparation of multi-gene-edited cell lines suitable for xenotransplantation is provided. This application allows for the generation of cell lines lacking three major antigens (GGTA1, CMAH, β4GALNT2) while efficiently expressing seven human regulatory factors (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, EPCR). These cell lines can be used to prepare gene-edited pigs, which, when used as donors for xenotransplantation, can reduce the recipient's immune rejection of the xenotransplanted organ, significantly improving the survival time and physiological function continuity of the xenotransplanted organ in the recipient, thus providing research materials and new methods for xenotransplantation applications.

[0011] According to a third aspect of the present invention, an application of a multi-gene editing system in the preparation of multi-gene-edited animals suitable for xenotransplantation is provided. Thus, by using the prepared multi-gene-edited animal as a donor for xenotransplantation, the recipient's immune rejection of the xenogeneic organ can be reduced, significantly improving the survival time and physiological function continuity of the xenogeneic organ in the recipient's body, providing research materials and new methods for the application of xenotransplantation.

[0012] According to a fourth aspect of the present invention, an application of a multi-gene editing system in xenotransplantation is provided. This can significantly reduce the intensity of recipient immune rejection of xenotransplanted organs and improve the survival time and physiological function continuity of xenotransplanted organs within the recipient body.

[0013] According to a fifth aspect of the present invention, a multi-gene-edited cell line prepared by a multi-gene-editing system is provided. Thus, the multi-gene-edited cell line prepared by this system lacks three major antigens (GGTA1, CMAH, β4GALNT2) and efficiently expresses seven human regulatory factors (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, EPCR). Furthermore, by preparing multi-gene-edited pigs from this cell line, which can be used as a donor source for xenotransplantation, the intensity of immune rejection of xenotransplanted organs can be greatly reduced, and the survival time and physiological function persistence of xenotransplanted organs in the recipient body can be improved.

[0014] According to a sixth aspect of the present invention, a multi-gene-edited pig prepared using a multi-gene-editing system is provided. Therefore, by using this multi-gene-edited pig as a donor source for xenotransplantation, the intensity of immune rejection of the xenotransplanted organ by the recipient can be greatly reduced, and the survival time and physiological function continuity of the xenotransplanted organ in the recipient's body can be improved.

[0015] According to a seventh aspect of the present invention, an application of a multi-gene-edited cell line or multi-gene-edited pig in xenotransplantation is provided. This can significantly reduce the intensity of immune rejection of the xenogeneic organ by the recipient, and improve the survival time and physiological function continuity of the xenogeneic organ in the recipient's body.

[0016] According to an eighth aspect of the present invention, a multi-gene editing cell line is provided, the cell line being... Sus scrofaYZ10, with accession number GDMCC No: 68094, is a cell line that lacks three major antigens (GGTA1, CMAH, β4GALNT2) while efficiently expressing seven human regulatory factors (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, EPCR). This multi-gene-edited cell line can then be further processed into multi-gene-edited pigs, which, as donors for xenotransplantation, can significantly reduce the recipient's immune rejection of xenotransplanted organs and improve the survival time and physiological function of xenotransplanted organs in the recipient.

[0017] According to a ninth aspect of the present invention, a method for preparing a multi-gene-edited cell line is provided, the method comprising the following steps: S1: Synthesize the sequences shown in SEQ ID NO:41 and SEQ ID NO:43, and then clone the synthesized sequences into the pUC57 vector to obtain expression vector H11 pUC57 and expression vector Rosa26 PUC57; or directly synthesize the expression vector H11 pUC57 and expression vector Rosa26 PUC57. S2: Pig cells were transfected with expression vectors H11 pUC57 and Rosa26 pUC57. Cell lines were then selected to obtain positive cell lines that simultaneously knocked out three genes (GGTA1, CMAH, and β4GALNT2) and site-specifically integrated seven genes (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, and EPCR). This method allows for the preparation of multi-gene-edited cell lines that lack three major antigens (GGTA1, CMAH, and β4GALNT2) while efficiently expressing seven human regulatory factors (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, and EPCR). These multi-gene-edited cell lines can then be further processed into multi-gene-edited pigs, which, as donors for xenotransplantation, can significantly reduce the recipient's immune rejection of xenotransplanted organs and improve the survival time and physiological function continuity of xenotransplanted organs in the recipient body.

[0018] According to a tenth aspect of the present invention, a multi-gene-edited cell line prepared by the aforementioned method is provided. Thus, the multi-gene-edited cell line prepared by this method lacks three major antigens (GGTA1, CMAH, β4GALNT2) while efficiently expressing seven human regulatory factors (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, EPCR). This multi-gene-edited cell line can then be further processed into multi-gene-edited pigs, which, as donor sources for xenotransplantation, can significantly reduce the recipient's immune rejection of xenotransplanted organs and improve the survival time and physiological function continuity of xenotransplanted organs in the recipient body.

[0019] According to an eleventh aspect of the present invention, an application of a cell line in the preparation of multi-gene-edited pigs is provided. Therefore, by using this multi-gene-edited pig as a donor source for xenotransplantation, the intensity of immune rejection of the xenotransplanted organ by the recipient can be greatly reduced, and the survival time and physiological function persistence of the xenotransplanted organ in the recipient's body can be improved.

[0020] According to a twelfth aspect of the present invention, a cell line is provided for use in the preparation of multi-gene-edited pigs for xenotransplantation. This significantly reduces the intensity of immune rejection of the xenogeneic organ by the recipient, and improves the survival time and physiological function continuity of the xenogeneic organ in the recipient.

[0021] The beneficial effects of this invention are as follows: Addressing the industry pain points of existing research, which often focuses on single-gene modification, involves lengthy and complex construction processes, and suffers from insufficient genetic stability and reproducibility of models, this invention utilizes optimized multi-gene editing technology and systems to achieve efficient integration and stable expression of the deletion of three major antigens (GGTA1, CMAH, β4GALNT2) and seven human regulatory factors (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, EPCR), resulting in cell line 45. Genetically modified pigs prepared using this cell line model can serve as xenotransplant donor pigs. These donor pigs significantly reduce the recipient's immune rejection of xenotransplant organs, greatly enhancing the survival time and physiological function persistence of xenotransplant organs in the recipient body. This provides a standardized and systematic key technical pathway for the translation of xenotransplantation from basic experiments to clinical applications. Attached Figure Description

[0022] Figure 1 Map of the pKLV2-U6gRNA5(BbsI)-PGKpuro2AZsG-W expression vector; Figure 2 A schematic diagram of the constructed H11 site multi-gene integrated expression cassette structure; Figure 3The constructed H11 PUC57 vector map; Figure 4 A schematic diagram of the constructed Rosa26 site multi-gene integrated expression cassette structure; Figure 5 To construct the ROSA26 PUC57 vector map; Figure 6 Results of hCD39 and hCD46 detection; Figure 7 Results of hCD47 and hCD55 detection; Figure 8 Results of hCD59 and EPCR detection; Figure 9 The results of hTBM detection; Figure 10 Image of a monoclonal cell; Figure 11 This is a diagram showing the sequence alignment results of the H11 gene locus. Figure 12 This is a diagram showing the sequence alignment results of the Rosa26 gene locus. Figure 13 This is a diagram showing the sequence alignment results of the GGTA1 gene. Figure 14 This is a diagram showing the CMAH gene sequence alignment results; Figure 15 This is a diagram showing the sequence alignment results of the β4GALNT2 gene. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] Example 1: Selection of target genes for editing.

[0026] 1.1 Selection of target genes for knockout.

[0027] The GGTA1 gene is involved in the synthesis of α-Gal antigen, which has widely present natural antibodies against in the human body; the CMAH gene can cause pig cells to produce sialic acid structures that are not present in the human body; and the β4GALNT2 gene is involved in the synthesis of another type of carbohydrate antigen closely related to immune rejection. These three types of antigens work together during xenotransplantation to significantly enhance the human immune system's rejection response to pig organs.

[0028] However, knocking out only one gene often fails to fundamentally reduce the level of immune rejection. Therefore, this application designs the simultaneous knockout of three key genes: GGTA1, CMAH, and β4GALNT2, to more comprehensively reduce the immunogenicity on the surface of porcine cells, thereby providing a more favorable foundation for xenotransplantation.

[0029] 1.2 Selection of knock-in target genes.

[0030] In the field of xenotransplantation research, simply reducing immune rejection through gene knockout still has certain limitations. To further enhance the biocompatibility and physiological stability of pig organs in the recipient environment, specific humanized functional genes can be directionally knocked into the pig genome, making them more similar to humans in immune regulation and physiological metabolic mechanisms, which can further improve the success rate of xenotransplantation.

[0031] Among numerous humanized immunomodulatory molecules, hCD46, hCD55, and hCD59 act at different key nodes in the complement activation cascade, significantly inhibiting complement-mediated cell lysis and tissue damage by constructing a complete complement inhibition barrier on the donor cell surface. Simultaneously, the protein C anticoagulation pathway, composed of hTBM and EPCR, plays a central role in regulating coagulation homeostasis, suppressing inflammatory responses, and maintaining vascular endothelial integrity, effectively preventing the risk of post-transplant thrombosis. Furthermore, hCD39 further blocks platelet activation and the inflammatory cascade by hydrolyzing extracellular nucleotides with pro-inflammatory and procoagulant effects; while hCD47 significantly reduces the probability of donor cells being recognized and cleared by macrophages by transmitting "self" signals (inhibitory signals) to recipient innate immune cells.

[0032] Based on the aforementioned multiple protective mechanisms, integrating various functional genes such as hCD46, hCD55, hCD59, hTBM, EPCR, hCD39, and hCD47 into the donor cell or animal genome can create a synergistic protective effect across multiple dimensions, including complement inhibition, anticoagulation, anti-inflammation, and immune evasion. This "multi-target, comprehensive" gene modification strategy will significantly improve the survival stability and functional continuity of xenotransplanted organs in the recipient body.

[0033] Example 2: Construction and efficiency verification of sgRNA expression vector.

[0034] 2.1 sgRNA sequence design.

[0035] To construct a gene-edited pig model that simultaneously possesses the deletion of three core xenogeneic antigens (GGTA1, CMAH, β4GALNT2) and the steady-state expression of seven humanized functional genes (hCD46, hCD55, hCD59, hTBM, EPCR, hCD39, and hCD47), this invention designs and optimizes specific sgRNAs for knockout targets (GGTA1, CMAH, β4GALNT2) and exogenous gene integration targets (H11, Rosa26).

[0036] 2.11 Design of sgRNA sequences targeting knockout sites.

[0037] Based on the gRNA of exon 7 of the CMAH gene (CMAH cytidine monophospho-N-acetylneuraminic acid hydroxylase [Sus scrofa (pig)] Gene ID: 396918) and the gRNA of exon 3 of the GGTA1 gene (glycoprotein alpha-galactosyltransferase 1 [Sus scrofa (pig)] Gene ID: 396733), and targeting exons 3, 4, and 6 of the β4GALNT2 gene (beta-1,4-N-acetyl-galactosaminyltransferase 2 (SID blood group) [Sus scrofa (pig)] Gene ID: 100621328), six gRNAs (β4GALNT2 sg3) were designed. The sequences of these gRNAs are shown in Table 1.

[0038] Table 1. sgRNA sequences of GGTA1, CMAH, and β4GALNT2 genes CMAH sgRNA GAGTAAGGTACGTGATCTGT SEQ ID NO:1 GGTA1 sgRNA GAGAAAATAATGAATGTCAA SEQ ID NO:2 B4GALNT2 sgRNA1 TTGAGGATCGACAGACATCT SEQ ID NO:3 B4GALNT2 sgRNA2 GGGACGGGATGGGTGAGTTG SEQ ID NO:4 B4GALNT2 sgRNA3 GAGGCATCACTTCCACCCCG SEQ ID NO:5 B4GALNT2 sgRNA4 AGGAAAGCTATAACTTGG SEQ ID NO:6 B4GALNT2 sgRNA5 CTGTATCGAGGAACACGCTT SEQ ID NO:7 B4GALNT2 sgRNA6 GTCTCCTCAGGTTCACTGCG SEQ ID NO:8 2.12 Design of sgRNA sequences targeting the knock-in site.

[0039] To achieve site-specific, stable, and efficient expression of human immune and coagulation regulatory gene clusters in the porcine genome, this invention selected the porcine endogenous safe harbor loci H11 and Rosa26 as integration targets for exogenous genes. Since the cleavage efficiency of the safe harbor loci directly affects the homologous recombination efficiency of large donor DNA fragments, this invention designed and screened specific sgRNAs with high cleavage activity and low off-target effects for these two loci, their sequences of which are shown in Table 2.

[0040] Table 2. sgRNA sequences at H11 and Rosa26 sites. H11 sgRNA1 GTTCCTGGAAGTTTAGATCA SEQ ID NO:9 H11 sgRNA2 AGATCAGGGTGGGCAGCTCT SEQ ID NO:10 ROSA26 sgRNA1 CCAAGAATCAGGTTAAGCCA SEQ ID NO:11 ROSA26 sgRNA2 CGAGAAGGAGCAAACTGACA SEQ ID NO:12 ROSA26 sgRNA3 GAAGGAGCAAACTGACATGG SEQ ID NO:13 2. Synthesis of 2 sgRNA oligonucleotide sequences.

[0041] Based on the sgRNA sequences in Tables 1 and 2, the corresponding annealing primers were designed as shown in Table 3.

[0042] Add 5 μL each of the upstream and downstream primers of the sgRNA from Table 3 to a 200 μL centrifuge tube and mix thoroughly by pipetting. Anneal the mixture: 95℃ for 10 min; 65℃ for 30 min. Dilute the annealed sgRNA product 10-fold with 90 μL ddH2O for later use.

[0043] Table 3 sgRNA annealing primer sequences CMAH sgRNA-F caccgGAGTAAGGTACGTGATCTGT SEQ ID NO:14 CMAH sgRNA-R aaacACAGATCACGTACCTTACTCc SEQ ID NO:15 GGTA1 sgRNA-F caccgGAGAAAATAATGAATGTCAA SEQ ID NO:16 GGTA1 sgRNA-R aaacTTGACATTCATTATTTTCTCc SEQ ID NO:17 B4GALNT2 sgRNA-1F caccg TTGAGGATCGACAGACATCT SEQ ID NO:18 B4GALNT2 sgRNA-1R aaac AGATGTCTGTCGATCCTCAAc SEQ ID NO:19 B4GALNT2 sgRNA-2F caccg GGGACGGGATGGGTGAGTTG SEQ ID NO:20 B4GALNT2 sgRNA-2R aaac CAACTCACCCATCCCGTCCCc SEQ ID NO:21 B4GALNT2 sgRNA-3F caccgGAGGCATCACTTCCACCCCG SEQ ID NO:22 B4GALNT2 sgRNA-3R aaacCGGGGTGGAAGTGATGCCTCc SEQ ID NO:23 B4GALNT2 sgRNA-4F caccgAGGAAAGCTATAACTTGG SEQ ID NO:24 B4GALNT2 sgRNA-4R aaacCCAAGTTATAGCTTTCCTc SEQ ID NO:25 B4GALNT2 sgRNA-5F caccgCTGTATCGAGGAACACGCTT SEQ ID NO:26 B4GALNT2 sgRNA-5R aaacAAGCGTGTTCCTCGATACAGc SEQ ID NO:27 B4GALNT2 sgRNA-6F caccgGTCTCCTCAGGTTCACTGCG SEQ ID NO:28 B4GALNT2 sgRNA-6R aaacCGCAGTGAACCTGAGGAGACc SEQ ID NO:29 H11 sgRNA-1F caccg GTTCCTGGAAGTTTAGATCA SEQ ID NO:30 H11 sgRNA-1R aaac TGATCTAAACTTCCAGGAACc SEQ ID NO:31 H11 sgRNA-2F caccg AGATCAGGGTGGGCAGCTCT SEQ ID NO:32 H11 sgRNA-2R aaacAGAGCTGCCCACCCTGATCTc SEQ ID NO:33 ROSA26 sgRNA-1F caccgCCAAGAATCAGGTTAAGCCA SEQ ID NO:34 ROSA26 sgRNA-1R aaacTGGCTTAACCTGATTCTTGGc SEQ ID NO:35 ROSA26 sgRNA-2F caccgCGAGAAGGAGCAAACTGACA SEQ ID NO:36 ROSA26 sgRNA-2R aaacTGTCAGTTTGCTCCTTCTCGc SEQ ID NO:37 ROSA26 sgRNA-3F caccgGAAGGAGCAAACTGACATGG SEQ ID NO:38 ROSA26 sgRNA-3R aaacCCATGTCAGTTTGCTCCTTCc SEQ ID NO:39 2.3 Connection and construction of expression vectors.

[0044] pKLV2-U6gRNA5(BbsI)-PGKpuro2AZsG-W (purchased from Addgene: 67975) was selected as the sgRNA expression vector, and its vector map is shown below. Figure 1 As shown in Table 4, the vector backbone was digested using Bbs1 enzyme. The digestion system was as shown in Table 4 below. After digestion in a metal bath at 37°C for 3 hours, the product was recovered to obtain the digested product.

[0045] Table 4 Enzyme digestion system pKLV2-U6gRNA5(BbsI)-PGKpuro2AZsG-W plasmid 3 μg 10×rCutSmart Buffer 5 μL Bbs1-HF 1 μL <![CDATA[H2O]]> up to 50 μL

[0046] The annealing product obtained in step 2.2 was ligated to the backbone of the sgRNA expression vector (pKLV2-U6gRNA5(BbsI)-PGKpuro2AZsG-W) using DNA ligase, as shown in Table 5 below. After mixing all components, the mixture was incubated at 25°C for 10 min in a constant temperature metal bath. After ligation, the mixture was added to competent cells, followed by a 5-min ice bath, a 45-s water bath at 42°C, and a 2-min ice bath. The cells were then plated onto solid culture medium for transformation. After overnight incubation, appropriately sized single colonies were picked and placed in 1.5 ml centrifuge tubes, and 700 μL of LB liquid medium was added. The cells were cultured at 37°C and 220 rpm for 8 h on a shaker. Sequencing was performed to confirm successful vector construction before scaling up the culture and extracting the sgRNA expression vector plasmid.

[0047] Table 5 Connection System sgRNA expression vector backbone 50 ng annealed product 1 μL DNA ligation mix 5 μL <![CDATA[H2O]]> up to 10 μL Example 3: Cellular experiments to verify the editing efficiency of different sgRNAs.

[0048] When the PK15 cell density in 10 cm culture dishes reached 90%–100%, they were digested and collected. The constructed sgRNA expression vector and Cas9 expression plasmid (purchased from Addgene: 62988) (5 μg each) were co-electroplated into PK15 cells at a ratio of 10 μg total plasmid per 1 / 3 of the dish. After 72 hours, flow cytometry was performed to sort the cells based on green fluorescent protein, obtaining a pool of cells exhibiting green fluorescence. The genomic DNA was extracted and amplified by PCR. The amplified products were sent to the company for sequencing, and the editing efficiency of the sgRNA was analyzed using the online website https: / / ice.editco.bio / # / . Finally, the sgRNAs with the best activity at the H11, ROSA26, and β4GALNT2 gene loci were selected for subsequent multi-gene editing experiments. The final selected sgRNAs with the best activity are shown in Table 6 below: Table 6. List of effective sgRNA sequences after screening. β4GALNT2 sgRNA3 GAGGCATCACTTCCACCCCG H11 sgRNA1 GTTCCTGGAAGTTTAGATCA ROSA26 sgRNA3 GAAGGAGCAAACTGACATGG GGTA1 sgRNA GAGAAAATAATGAATGTCAA CMAH sgRNA1 GAGTAAGGTACGTGATCTGT Example 4: Construct two vectors containing a knock-in gene and a tandem triple gene KO.

[0049] Both Rosa26 and H11 sites are recognized as gene safe harbor sites, which can be used for the targeted integration of exogenous genes and can ensure the stable expression of the introduced gene in the host genome.

[0050] Genes to be knocked in: Human CD39 gene hCD39 (ENTPD1) (ectonucleoside triphosphate diphosphohydrolase 1 [Homo sapiens(human)] Gene ID: 953; CDS GenBank accession number: NM_001776.6); Human membrane cofactor protein gene hCD46 (CD46 molecule [Homo sapiens(human)] NCBI Gene ID: 4179; CDS GenBank accession number: NM_002389.4); Human CD47 gene hCD47 (CD47 molecule [Homo sapiens(human)] NCBI Gene ID: 961; CDS GenBank accession number: NM_001777.4); Human decay accelerator gene hCD55 (CD55 molecule (Cromer blood group) [Homo sapiens(human)] NCBI Gene ID: 1604; CDS GenBank accession number: NM_000574.5); human endothelial protein C receptor gene EPCR (also known as PROCR) (protein C receptor [Homo sapiens(human)] NCBI Gene ID: 10544; CDSG GenBank accession number: NM_006404.5); human thrombomodulin gene hTBM (THBD) (thrombomodulin [Homo sapiens(human)]; NCBI Gene ID: 7056; CDS GenBank accession number: NM_000361.3); human complement regulatory protein gene hCD59 (CD59molecule (CD59blood group) [Homo sapiens(human)]; NCBI Gene ID: 966; CDS GenBank accession number: NM_001127223.1).

[0051] 4.1 Construction of H11 PUC57 plasmid.

[0052] The H11 PUC57 plasmid uses the porcine H11 site as the target integration site to specifically integrate the hCD39, hCD46, hCD47, and hCD55 genes.

[0053] This invention screened numerous arrangements of sgRNA sequences for H11, Rosa26 (integration site), β4GALNT2, GGTA1, and CMAH (knockout site), selecting the optimal, shortest, and most efficient arrangement and connection method. A U6 promoter was then added at the beginning to obtain a multi-gene editing fragment, the sequence of which is as follows (SEQ ID NO:40): GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC GAGAAAATAATGAATGTCAA GTTTAAGAGCTATGCTGGAAACAGCAT AGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC TTTTTTTGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC GAGTAAGGTACGTGATCTGT GTTTAA GAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGC TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC TTTTTTTGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAGGCAT CACTTCCACCCCG GTTTAA GAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGC TTTTTTTGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC GTTCCTGGAAGTTTAGATCA GTTTAA GAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC GGTGC TTTTTTTGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC GAAGGAGCAAACTGACATGG GTTTAA GAGCTATGCTGGAAACA GCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC TTTTTTT.

[0054] Note: In this sequence, the regular font indicates the U6 promoter; the underlined text indicates sgRNA; and the italicized and bold text indicates scaffold. The scaffold sequence is an important component of sgRNA in the CRISPR-Cas system. Its main function is to bind to Cas proteins and stabilize the secondary structure of sgRNA, thereby promoting the recognition and binding of sgRNA to target DNA.

[0055] The CDS regions of the four genes to be knocked in (hCD39, hCD46, hCD47, and hCD55) were synthesized. The foreign genes were arranged in the order hCD39, hCD46, hCD47, and hCD55, and linked by a self-cleaved peptide sequence (T2A) to allow multiple foreign genes to be expressed in the same transcript. A termination signal was added after each foreign gene expression unit to ensure normal transcription termination and stable expression. Downstream of this multi-gene expression frame, a NeoR (neomycin resistance gene) selection marker was further linked for subsequent positive clone screening in mammalian cells. Subsequently, this multi-gene expression frame was tandemly linked with a U6 promoter-driven sgRNA expression module to form a complex fragment. To achieve stable integration of the foreign fragment into the genome, left and right homologous arm sequences were designed and introduced on both sides of the complex fragment based on the porcine H11 site sequence. Homologous recombination was used to guide the site-specific integration of the foreign gene at the H11 site, as shown in the schematic diagram below. Figure 2 As shown, its nucleotide sequence is as follows (SEQ ID NO:41): aactccctcttaccaacttattactactaacttcccaagtactggctgctcagctgcttccttgggca tgggggagggagcactattttttcctctcctgacttcatcctcttccttttaatttccataaggttccctgtggcc ctgtgcttttttattttgaggccttgcacatccttctggccctgattgcttctcaactcatcttgtgcctgctgga cttccaccgttgtttcatgtatctcgttagctgagatagcacttcctcctgcccttaccctttatctggctcttag ctcctaaaaactgcattattagcttcctcttttgcctctactcttactcaaccaaaattgttttaagatctgtgga tctagcttctgctgtgctattcttaggaacacttttatttcctcttagctccatctcaccagttattggctaatgg ctttgcttggtacctacatctgtacatttctttcgtactagcttctagactgaaaaaggactgttggttcaacatg aaagggaaggaggtaaaagaggacacacaggaaagatggattgggattcaggtctctgctgttgttacttgagatt gctttctagattctacttgtggaaacaaaaagcctttgcgagaattctaaactggagtatttctgtaattgaggag tcttgctcagcaaatcccacttaggggactaatgaagtaccaggaagagacagaccatgctcaatccacaaagcca ggttttactgaaatgtgacctactttcttatgttcctggaagtttaga CTAGCTAGCTAGGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGCATCCATCTAGATCGCCACC ATGGAAGATACAAAGGAGT CTAACGTGAAGACATTTTGCTCCAAGAATATCCTAGCCATCCTTGGCTTCTCCTCTATCATAGCTGTGATAGCTTT GCTTGCTGTGGGGTTGACCCAGAACAAAGCATTGCCAGAAAACGTTAAGTATGGGATTGTGCTGGATGCGGGTTCT TCTCACACAAGTTTATACATCTATAAGTGGCCAGCAGAAAAGGAGAATGACACAGGCGTGGTGCATCAAGTAGAAG AATGCAGGGTTAAAGGTCCTGGAATCTCAAAATTTGTTCAGAAAGTAAATGAAATAGGCATTTACCTGACTGATTG CATGGAAAGAGCTAGGGAAGTGATTCCAAGGTCCCAGCACCAAGAGACACCCGTTTACCTGGGAGCCACGGCAGGC ATGCGGTTGCTCAGGATGGAAAGTGAAGAGTTGGCAGACAGGGTTCTGGATGTGGTGGAGAGGAGCCTCAGCAACT ACCCCTTTGACTTCCAGGGTGCCAGGATCATTACTGGCCAAGAGGAAGGTGCCTATGGCTGGATTACTATCAACTA TCTGCTGGGCAAATTCAGTCAGAAAACAAGGTGGTTCAGCATAGTCCCATATGAAACCAATAATCAGGAAACCTTT GGAGCTTTGGACCTTGGGGGAGCCTCTACACAAGTCACTTTTGTACCCCAAAACCAGACTATCGAGTCCCCAGATA ATGCTCTGCAATTTCGCCTCTATGGCAAGGACTACAATGTCTACACACATAGCTTCTTGTGCTATGGGAAGGATCA GGCACTCTGGCAGAAACTGGCCAAGGACATTCAGGTTGCAAGTAATGAAATTCTCAGGGACCCATGCTTTCATCCT GGATATAAGAAGGTAGTGAACGTAAGTGACCTTTACAAGACCCCCTGCACCAAGAGATTTGAGATGACTCTTCCAT TCCAGCAGTTTGAAATCCAGGGTATTGGAAACTATCAACAATGCCATCAAAGCATCCTGGAGCTCTTCAACACCAG TTACTGCCCTTACTCCCAGTGTGCCTTCAATGGGATTTTCTTGCCACCACTCCAGGGGGATTTTGGGGCATTTTCA GCTTTTTACTTTGTGATGAAGTTTTTAAACTTGACATCAGAGAAAGTCTCTCAGGAAAAGGTGACTGAGATGATGA AAAAGTTCTGTGCTCAGCCTTGGGAGGAGATAAAAACATCTTACGCTGGAGTAAAGGAGAAGTACCTGAGTGAATA CTGCTTTTCTGGTACCTACATTCTCTCCCTCCTTCTGCAAGGCTATCATTTCACAGCTGATTCCTGGGAGCACATC CATTTCATTGGCAAGATCCAGGGCAGCGACGCCGGCTGGACTTTGGGCTACATGCTGAACCTGACCAACATGATCC CAGCTGAGCAACCATTGTCCACACCTCTCTCCCACTCCACCTATGTCTTCCTCATGGTTCTATTCTCCCTGGTCCT TTTCACAGTGGCCATCATAGGCTTGCTTATCTTTCACAAGCCTTCATATTTCTGGAAAGATATGGTA GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCG ATGGAGCCTCCCGGCCGCCGCGAGTGTC CCTTTCCTTCCTGGCGCTTTCCTGGGTTGCTTCTGGCGGCCATGGTGTTGCTGCTGTACTCCTTCTCCGATGCCTG TGAGGAGCCACCAACATTTGAAGCTATGGAGCTCATTGGTAAACCAAAACCCTACTATGAGATTGGTGAACGAGTA GATTATAAGTGTAAAAAAGGATACTTCTATATACCTCCTCTTGCCACCCATACTATTTGTGATCGGAATCATACAT GGCTACCTGTCTCAGATGACGCCTGTTATAGAGAAACATGTCCATATATACGGGATCCTTTAAATGGCCAAGCAGT CCCTGCAAATGGGACTTACGAGTTTGGTTATCAGATGCACTTTATTTGTAATGAGGGTTATTACTTAATTGGTGAA GAAATTCTATATTGTGAACTTAAAGGATCAGTAGCAATTTGGAGCGGTAAGCCCCCAATATGTGAAAAGGTTTTGT GTACACCACCTCCAAAAATAAAAAATGGAAAACACACCTTTAGTGAAGTAGAAGTATTTGAGTATCTTGATGCAGT AACTTATAGTTGTGATCCTGCACCTGGACCAGATCCATTTTCACTTATTGGAGAGAGCACGATTTATTGTGGTGAC AATTCAGTGTGGAGTCGTGCTGCTCCAGAGTGTAAAGTGGTCAAATGTCGATTTCCAGTAGTCGAAAATGGAAAAC AGATATCAGGATTTGGAAAAAATTTTACTACAAAGCAACAGTTATGTTTGAATGCGATAAGGGTTTTTACCTCGA TGGCAGCGACACAATTGTCTGTGACAGTAACAGTACTTGGGATCCCCCAGTTCCAAGTGTCTTAAAGTGCTGCCT CCATCTAGTACAAAACCTCCAGCTTTGAGTCATTCAGTGTCGACTTCTTCCACTACAAAATCTCCAGCGTCCAGTG CCTCAGGTCCTAGGCCTACTTACAAGCCTCCAGTCTCAAATTATCCAGGATATCCTAAACCTGAGGAAGGAATACT TGACAGTTTGGATGTTTGGGTCATTGCTGTGATTGTTATTGCCATAGTTGTTGGAGTTGCAGTAATTTGTGTTGTC CCGTACAGATATCTTCAAAGGAGGAAGAAGAAAGGCACATACCTAACTGATGAGACCCACAGAGAAGTAAATTTTA CTTCTCTCTGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGTGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGG GATGCGGTGGGCTCTATGGCGTACGGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGT ACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCCACCCCAATTTTGTATTTATTTTTTTAATTATT TGTGCAGCGATGGGGCGGGGGGGGGGGGGGCGGCGCCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTCCTTTTATGGCGAGCGGCGGCGGCGGCGCCTATAAAAAGCGAAGCGGCCGGCGGCGCATCCATCTAGATCGCCACCC ATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCGTGCTGCGGATCAGCTCAGCTACTATT TAATAAAACAAAATCTGTAGAATTCACGTTTTGTAATGACACTGTCGTCATTCCATGCTTTGTTACTAATATGGAG GCACAAAACACTACTGAAGTATACGTAAAGTGGAAATTTAAAGGAAGATTTTACACCTTTGATGGAGCTCTAA ACAAGTCCACTGTCCCCACTGACTTTAGTAGTGCAAAA TO GAAGTCTCACAATTACTAAAAGGAGATGCCTCTTT GAAGATGGATAAGAGTGATGCTGTCTCACACAGGAAACTACACTTGTGAAGTAACAGAATTAACCAGAGAAGGT GAAACGATCATCGAGCTAAAATATCGTGTTGTTTCATGGTTTTCTCCAAATGAAAATATTCTTATTGTTATTTTCC CAATTTTTGCTATACTCCTGTTCTGGGGACAGTTTGGTATTAAAACACTTAAATAGATCCGGTGGTATGGATGA GAAAACAATTGCTTTACTTGTTGCTGGACTAGTGATCACTGTCATTGTCATTGTTGGAGCCATTCTTTTCGTCCCA GGTGAATATTCATTAAAGAATGCTACTGGCCTTGGTTTAATTGTGACTTCTACAGGGATATTAATATTACTTCACT ACTATGTGTTTAGTACAGCGATTGGATTAACCTCCTTCGTCATTGCCATATTGGTTATTCAGGTGATAGCCTATAT CCTCGCTGTGGTTGGACTGAGTCTCTGTATTGCGGCGTGTATACCAATGCATGGCCCTCTTCTGATTTCAGGTTTG AGTATCTTAGCTCTAGCACAATTACTTGGACTAGTTTATATGAAATTTGTGGCTTCCAATCAGAAGACTATACAAC CTCCTAGGAAAGCTGTAGAGGAACCCCTTAATGCATTCAAAGAATCAAAAGGAATGATGAATGATGAA GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCG ATGACCGTCGCGCGGCCGAGCGTGCCC GCGGCGCTGCCCCTCCTCGGGGAGCTGCCCCGGCTGCTGCTGCTGGTGCTGTTGTGCCTGCCGGCCGTGTGGGGTG ACTGTGGCCTTCCCCCAGATGTACCTAATGCCCAGCCAGCTTTGGAAGGCCGTACAAGTTTTCCCGAGGATACTGT AATAACGTACAAATGTGAAGAAAGCTTTGTGAAAATTCCTGGCGAGAAGGACTCAGTGATCTGCCTTAAGGGCAGT CAATGGTCAGATATTGAAGAGTTCTGCAATCGTAGCTGCGAGGTGCCAACAAGGCTAAATTCTGCATCCCTCAAAC AGCCTTATATCACTCAGAATTATTTTCCAGTCGGTACTGTTGTGGAATATGAGTGCCGTCCAGGTTACAGAAGAGA ACCTTCTCTATCACCAAAACTAACTTGCCTTCAGAATTTAAAATGGTCCACAGCAGTCGAATTTTGTAAAAAGAAA TCATGCCCTAATCCGGGAGAAATACGAAATGGTCAGATTGATGTACCAGGTGGCATATTATTTGGTGCAACCATCT CCTTCTCATGTAACACAGGGTACAAATTATTTGGCTCGACTTCTAGTTTTTGTCTTATTTCAGGCAGCTCTGTCCA GTGGAGTGACCCGTTGCCAGAGTGCAGAGAAATTTATTGTCCAGCACCACCACAAATTGACAATGGAATAATTCAA GGGGAACGTGACCATTATGGATATAGACAGTCTGTAACGTATGCATGTAATAAAGGATTCACCATGATTGGAGAGC ACTCTATTTATTGTACTGTGAATAATGATGAAGGAGAGTGGAGTGGCCCACCACCTGAATGCAGAGGAAAATCTCT AACTTCCAAGGTCCCACCAACAGTTCAGAAACCTACCACAGTAAATGTTCCAACTACAGAAGTCTCACCAACTTCT CAGAAAACCACCACAAAAACCACCACACCAAATGCTCAAGCAACACGGAGTACACCTGTTTCCAGGACAACCAAGC ATTTTCATGAAACAACCCCAAATAAAGGAAGTGGAACCACTTCAGGTACTACCCGTCTTCTATCTGGGCACACGTG TTTCACGTTGACAGGTTTGCTTGGGACGCTAGTAACCATGGGCTTGCTGACTTAG CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGataacttcgtatagcatacattatacgaagttat TTGGCGCGCCAAGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCA ATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCC CGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGA ACACAGGACCGGTTCTAGAGCGCTGCCACCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGG TGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGC GCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGG CTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGC TGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGC TGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAG CGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAG CCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTT GCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTAT CAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTT ACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTG GGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAA GGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTT CGCCCACCCCCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAG GTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCT GGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGC TCTATGGATAAGAATGCGGCCGCTAAACTATACGCGTtcagg gtgggcagctctgggttttataggctacactgttaacactcaggctgttttctaccgtttagtcaaaatatagtca ccttgcctgcttcacctgtccatcagagaatggcctcattaattgactctctagtatgaagtcaaagtagctttgg tggccctaaatggacaagtatcaagagactgggtgaattgaggagcttgagactgtcacctcagatcgaaaagact gaaaaatcacctcagatcaaaaagactgaaaaatcttcagtctggaaaggggactcaaaaccataattagagtatt ctggtagaatccttttctccactgttattcatacagttaaggtgaataactaaaagtaattgtgagctgaggagta agatacaacacacaaggaatcagttaacagagtctcgagtgaaattataaatggaaagaattatgacttgaatcat aactctgaggccccattttccctaacaacttttgtcccaataaacgtgggtatttgtttgggagaaactatcatat acatgattacccagtaaacagactgtttactaagtgggtttaattttagaaattgcgcgctgcaatctggtattaa ccatacaactacctacctatagggtcagcccagcctgaactatcccattggggtctttattaaggctcaagaaacg gccatagcttcttcctttaaaatgagtgtttatttctatgagctttaaagaaaaaaacagataatttccctcaacc tactgaagaggaagggattcaggaagaaataaaca。

[0056] Note: In this sequence, the lowercase letters with underlined text represent homologous arm sequences; the uppercase letters with underlined text represent the CDS sequences of hCD39, hCD46, hCD47, and hCD55, respectively; the italicized and bold text represents the neomycin resistance gene sequence; and the ordinary lowercase letters represent the above-mentioned U6 tandem sg sequence.

[0057] The full-length sequence was synthesized by GenScript Biotech Co., Ltd. The synthesized full-length sequence, containing homologous arms, multiple gene expression cassettes, and a U6 tandem sgRNA module, was cloned into the commercial pUC57 vector (purchased from GenScript: SD1176) using the EcoRV restriction site, resulting in a recombinant plasmid named H11pUC57. Sequencing confirmed that the cloned insert sequence was completely identical to the designed sequence, as shown in the results below. Figure 3 The complete sequence of plasmid H11pUC57 is shown below (SEQ ID NO:42):

[0058] Note: Uppercase letters represent target sequences including CDS sequences of hCD39, hCD46, hCD47, and hCD55; lowercase letters represent the PUC57 plasmid backbone sequence.

[0059] 4.2 Construction of ROSA26 PUC57 plasmid.

[0060] The ROSA26 PUC57 plasmid uses the porcine Rosa26 site as the target integration site to specifically integrate the hCD59, hTBM, and EPCR genes.

[0061] Human EPCR, hTBM, and hCD59 were selected as functional genes. After optimizing their CDS regions with porcine codons, they were tandemly linked to form a multi-gene expression framework. Stop codons (TAA / TGA) were deleted from the ends of all three genes, and they were ligated via P2A self-cleaving peptide sequences to achieve co-expression of multiple genes in the same transcript. A bGH poly(A) signal sequence was then attached to the ends to ensure transcriptional stability. Subsequently, an independent gene editing and screening module was constructed by introducing a second promoter from the EF-1α core promoter. This module sequentially linked the Cas9 protein coding sequence, the nuclear localization signal (NLS), and a BSD (blastcin) selection marker gene tandemly linked via a P2A sequence. This allowed the Cas9 protein to enter the nucleus and perform its editing function while simultaneously conferring resistance to blastcin to positive clones.

[0062] To achieve stable integration of the exogenous fragment into the genome, left and right homologous arm sequences were designed and introduced on both sides of the complex fragment based on the porcine ROSA26 site sequence. Homologous recombination was used to guide the site-specific integration of the exogenous gene at the ROSA26 site, forming a full-length recombinant nucleic acid sequence, as shown in the schematic diagram below. Figure 4 As shown, its sequence is as follows (SEQ ID NO:43): c acaaatggcgtgttttggttggagtaaagctcctgtcagttacagcctcgggagtgcgcagcctcccaggaactct cgcattgccccctgggtgggtaggtaggtggggtggagagagctgcacaggcgggcgctgtcggcctcctgcgggg ggaggggagggtcagtgaaagtggctcccgcgcgggcgtcctgccaccctcccctccgggggagtcggtttacccg ccgcctgctcggctttggtatctgattggctgctgaagtcctgggaacggccccttgttattggcttgggtcccaa atgagcgaaaccactacgcgagtcggcagggaggcggtctttggtacggccctccccgaggccagcgccgcagtgt ctggcccctcgcccctgcgcaacgtggcaggaagcgcgcgcaggaggcgggggcgggctgccgggccgaggcttct gggtggtggtgactgcgactccgccctgggcgtccgccgcctgaaggacgagactagctctacctgctctcggacc cgtgggggtggggggtggaggaaggagtggggggtcggtcctgctggcttgtgggtgggaggcgcatgttctccaa aaacccgcgcgagctgcaatcctgagggagctgcagtggaggaggcggagagaaggccgcacccttctccgcaggg ggaggggagtgccgcaatacctttatgggagttctctgctgcctccttttcctaaggaccgccctgggcctagaaa aatccctccctcccccgcgatctcgtcatcgcctcca TTGGCGCGCCAAGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGCATCCATCTAGATCGCCACC ATGTTGACAACATTGCTGCCGATACTGCTG CTGTCTGGCTGGGCCTTTTGTAGCCAAGACGCCTCAGATGGCCTCCAAAGACTTCATATGCTCCAGATCTCCTACT TCCGCGACCCCTATCACGTGTGGTACCAGGGCAACGCGTCGCTGGGGGGACACCTAACGCACGTGCTGGAAGGCCC AGACACCAACACCACGATCATTCAGCTGCAGCCCTTGCAGGAGCCCGAGAGCTGGGCGCGCACGCAGAGTGGCCTG CAGTCCTACCTGCTCCAGTTCCACGGCCTCGTGCGCCTGGTGCACCAGGAGCGGACCTTGGCCTTTCCTCTGACCA TCCGCTGCTTCCTGGGCTGTGAGCTGCCTCCCGAGGGCTCTAGAGCCCATGTCTTCTTCGAAGTGGCTGTGAATGG GAGCTCCTTTGTGAGTTTCCGGCCGGAGAGAGCCTTGTGGCAGGCAGACACCCAGGTCACCTCCGGAGTGGTCACC TTCACCCTGCAGCAGCTCAATGCCTACAACCGCACTCGGTATGAACTGCGGGAATTCCTGGAGGACACCTGTGTGC AGTATGTGCAGAAACATATTTCCGCGGAAAACACGAAAGGGAGCCAAACAAGCCGCTCCTACACTTCGCTGGTCCT GGGCGTCCTGGTGGGCAGTTTCATCATTGCTGGTGTGGCTGTAGGCATCTTCCTGTGCACAGGTGGACGGCGATGT GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCG ATGCTTGGGGTCCTGGTCC TTGGCGCGCTGGCCCTGGCCGGCCTGGGGTTCCCCGCACCCGCAGAGCCGCAGCCGGGTGGCAGCCAGTGCGTCGA GCACGACTGCTTCGCGCTCTACCCGGGCCCCGCGACCTTCCTCAATGCCAGTCAGATCTGCGACGGACTGCGGGGC CACCTAATGACAGTGCGCTCCTCGGTGGCTGCCGATGTCATTTCCTTGCTACTGAACGGCGACGGCGGCGTTGGCC GCCGGCGCCTCTGGATCGGCCTGCAGCTGCCACCCGGCTGCGGCGACCCCAAGCGCCTCGGGCCCCTGCGCGGCTT CCAGTGGGTTACGGGAGACAACAACACCAGCTATAGCAGGTGGGCACGGCTCGACCTCAATGGGGCTCCCCTCTGC GGCCCGTTGTGCGTCGCTGTCTCCGCTGCTGAGGCCACTGTGCCCAGCGAGCCGATCTGGGAGGAGCAGCAGTGCG AAGTGAAGGCCGATGGCTTCCTCTGCGAGTTCCACTTCCCAGCCACCTGCAGGCCACTGGCTGTGGAGCCCGGCGC CGCGGCTGCCGCCGTCTCGATCACCTACGGCACCCCGTTCGCGGCCCGCGGAGCGGACTTCCAGGCGCTGCCGGTG GGCAGCTCCGCCGCGGTGGCTCCCCTCGGCTTACAGCTAATGTGCACCGCGCCGCCCGGAGCGGTCCAGGGGCACT GGGCCAGGGAGGCGCCGGGCGCTTGGGACTGCAGCGTGGAGAACGGCGGCTGCGAGCACGCGTGCAATGCGATCCC TGGGGCTCCCCGCTGCCAGTGCCCAGCCGGCGCCGCCCTGCAGGCAGACGGGCGCTCCTGCACCGCATCCGCGACG CAGTCCTGCAACGACCTCTGCGAGCACTTCTGCGTTCCCAACCCCGACCAGCCGGGCTCCTACTCGTGCATGTGCG AGACCGGCTACCGGCTGGCGGCCGACCAACACCGGTGCGAGGACGTGGATGACTGCATACTGGAGCCCAGTCCGTG TCCGCAGCGCTGTGTCAACACACAGGGTGGCTTCGAGTGCCACTGCTACCCTAACTACGACCTGGTGGACGGCGAG TGTGTGGAGCCCGTGGACCCGTGCTTCAGAGCCAACTGCGAGTACCAGTGCCAGCCCCTGAACCAAACTAGCTACC TCTGCGTCTGCGCCGAGGGCTTCGCGCCCATTCCCCACGAGCCGCACAGGTGCCAGATGTTTTGCAACCAGACTGC CTGTCCAGCCGACTGCGACCCCAACACCCAGGCTAGCTGTGAGTGCCCTGAAGGCTACATCCTGGACGACGGTTTC ATCTGCACGGACATCGACGAGTGCGAAAACGGCGGCTTCTGCTCCGGGGTGTGCCACAACCTCCCCGGTACCTTCG AGTGCATCTGCGGGCCCGACTCGGCCCTTGCCCGCCACATTGGCACCGACTGTGACTCCGGCAAGGTGGACGGTGG CGACAGCGGCTCTGGCGAGCCCCCGCCCAGCCCGACGCCCGGCTCCACCTTGACTCCTCCGGCCGTGGGGCTCGTG CATTCGGGCTTGCTCATAGGCATCTCCATCGCGAGCCTGTGCCTGGTGGTGGCGCTTTTGGCGCTCCTCTGCCACC TGCGCAAGAAGCAGGGCGCCGCCAGGGCCAAGATGGAGTACAAGTGCGCGGCCCCTTCCAAGGAGGTAGTGCTGCA GCACGTGCGGACCGAGCGGACGCCGCAGAGACTC GAGGGCAGAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCA ATGGGAATCCAAGGAGGGTCTGTCCTGTTCGGGCTGCTGCTCGTCCTGGCTGTCTTCTGCCATT CAGGTCATAGCCTGCAGTGCTACAACTGTCCTAACCCAACTGCTGACTGCAAAACAGCCGTCAATTGTTCATCTGA TTTTGATGCGTGTCTCATTACCAAAGCTGGGTTACAAGTGTATAACAAGTGTTGGAAGTTTGAGCATTGCAATTTC AACGACGTCACAACCCGCTTGAGGGAAAATGAGCTAACGTACTACTGCTGCAAGAAGGACCTGTGTAACTTTAACG AACAGCTTGAAAATGGTGGGACATCCTTATCAGAGAAAACAGTTCTTCTGCTGGTGACTCCATTTCTGGCAGCAGC CTGGAGCCTTCATCCCTAA ATGGCCAAGCCTTTGT CTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGT CGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGT GCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATG AGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCAT AGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGC TAA CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGAAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC TGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGATAAGAATGCGGCCGCTAAACTATataacttcgtatagcatacattatacgaagttat tgtcagtttgctccttct cgattatgggcgggattcttttgccctggcttaacctgattcttgggcgttgtcctgcaggggattgagcaggtgt acgaggacgagcccaatttctctatattcccacagtcttgagtttgtgtcacaaaataattatagtggggtggaga tgggaaatgagtccaggcaacacctaagcctgattttatgcattgagactgcgtgttattactaaagatctttgtg tcgcaatttcctgatgaagggagataggttaaaaagcacggatctactgagttttacagtcatcccatttgtagac ttttgctacaccaccaaagtatagcatctgagattaaatattaatctccaaaccttaggccccctcacttgcatcc ttacggtcagataactctcactcatactttaagcccattttgtttgttgtacttgctcatccagtcccagtcccat tggctttctcctcacctgttttaggtagccagcaagtcatgaaatcagataagttccaccaccaattaacactacc catcttgagcataggcccaacagtgcatttattcctcatttactgatgttcgtgaatatttaccttgattttcatt tttttctttttcttaagctgggattttactcctgaccctattcacagtcagatgatcttgactaccactgcgattg gacctgaggttcagcaatactcccctttatgtcttttgaataaatctgtttgtattttcattagtt agtaactgagctcagttgccgt。

[0063] Note: In this sequence, lowercase letters with underlined text represent homologous arms; uppercase letters with underlined text represent the CDS sequences of EPCR (PROCR), hTBM, and hCD59, respectively; ordinary lowercase letters represent the cas9 gene sequence; and italicized bold text represents the blast fungicide resistance gene.

[0064] Finally, the complete recombinant full-length sequence, including homologous arms and all functional elements, was commissioned to Nanjing GenScript Biotech Co., Ltd. for whole-genome synthesis. The synthesized full-length sequence was cloned into the commercially available pUC57 vector using the EcoRV restriction site, resulting in a recombinant donor vector with completely identical sequence verification. This vector was named the Rosa26 PUC57 plasmid, and the results are as follows: Figure 5 As shown, the complete sequence of the Rosa26 PUC57 plasmid is as follows (SEQ ID NO:44):

[0065] Note: Uppercase letters represent target sequences containing elements such as EPCR (PROCR), hTBM, hCD59, and Cas9; lowercase letters represent PUC57 plasmid backbone sequences.

[0066] Example 5: Introduction and expression of recombinant plasmids in cells.

[0067] 5.1 Transfection of Bama fibroblasts with H11 PUC57 plasmid and ROSA26 PUC57 plasmid.

[0068] Bama pig PFF cells were revived in 10cm cell culture dishes and electroporated when the cell density reached 100%. At 100% cell density, cells were divided into two groups: an experimental group (H11 PUC57 and Rosa26 PUC57) and a control group. For the experimental groups (H11 PUC57 and Rosa26 PUC57), half a 10cm cell culture dish was used, and 5μg of each of the two plasmids (H11 PUC57 or Rosa26 PUC57 plasmid) was electroporated at 520V. For the control group, half a 10cm cell culture dish was used, and cells were electroporated directly at 520V without plasmids, serving as a negative control. After electroporation, cells were transferred to antibiotic-free medium. Cell morphology and density were observed the next day. If morphology was normal but density was too low, the cells were transferred to antibiotic-containing normal medium for expansion. After 3 days, cells were enriched using BLAST and G418 (BLAST concentration: 6 ug / ml, G418 concentration: 400 ug / ml). After 3 days, one-third of the cell density was collected to assess editing efficiency.

[0069] 5.2 Verify the efficiency of the cell pool after 3 days of drug treatment.

[0070] The knock-in of large fragments was verified using a method that amplifies both left and right linkers, i.e., primers were designed across homologous arms. The amplification products were subjected to agarose gel electrophoresis. The results showed that the cell pool group amplified bands with the correct band size, while the control group wild-type cells amplified no bands. Based on this, to further verify whether the multi-gene expression cassettes integrated into the genome have transcriptional activity, total RNA was extracted from the cell pool and reverse transcribed into cDNA. The expression levels of seven target genes, hCD46, hCD55, hCD59, hTBM, EPCR, hCD39, and hCD47, were quantitatively detected using RT-qPCR. The results are as follows: Figures 6-9 As shown, hCD46, hCD55, hCD59, hTBM, EPCR, hCD39, and hCD47 were successfully transcribed, and their mRNA expression was significantly enhanced. Figure 6The results are for hCD39 and hCD46 detection. Figure 7 The results are for hCD47 and hCD55 detection. Figure 8 The results are for hCD59 and EPCR detection. Figure 9 The results are for hTBM detection.

[0071] The above results demonstrate that the multi-gene integrated knock-in system constructed in this invention can be stably initiated at the porcine safe harbor site, guiding seven exogenous genes—hCD46, hCD55, hCD59, hTBM, EPCR, hCD39, and hCD47—to achieve significant transcriptional upregulation at the mRNA level, successfully achieving a transcriptional breakthrough from scratch.

[0072] Primers are designed based on the gene knockout site to amplify the target fragment at the knockout site. Sequencing is then performed to check for any base insertions or deletions, and to determine whether the gene knockout has been achieved.

[0073] Example 5: Screening of cell lines with 7 gene knock-in and 3 gene knockout.

[0074] After 3 days of drug treatment, the remaining cells were serially diluted to obtain 10cm cell culture dishes containing 4000 cells each. These were cultured in a medium with a high concentration of serum (20%, normal serum concentration is 10%), with the medium changed every 2 days. After 10 days, individual cells formed clusters, as shown in the results. Figure 10 As shown ( Figure 10 The left and right images show the cluster morphology of different monoclonal colonies after they have reached confluence. Cell clones were picked using a cloning loop and transferred to 48-well plates for culture. After confluence, half of the cells were used for genotyping, while the remaining cells were transferred to 24-well plates. Positive monoclonal cells were identified and designated as cell line 45. The results of cell line 45 showed that the target gene was successfully knocked in, and that GGTA1, CMAH, and β4GALNT2 genes were all homozygous knocked out (homozygous knockout: knockout of both chromosomes). The gene sequence alignment results of cell line 45 with the wild-type cell line are as follows: Figures 11-15 As shown: Among them, Figure 11 This is a diagram showing the sequence alignment results of the H11 gene locus. Figure 12 This is a diagram showing the sequence alignment results of the Rosa26 gene locus. Figure 13 This is a diagram showing the sequence alignment results of the GGTA1 gene. Figure 14 This is a diagram showing the CMAH gene sequence alignment results. Figure 15 This is a diagram showing the sequence alignment results of the β4GALNT2 gene.

[0075] This cell line No. 45 was preserved as a cell culture, and the preservation name is Sow YZ10, accession number GDMCCNo: 68094, classification name Pig The deposit date is April 16, 2026. The depository is Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0076] The gene knockout comparison results of the preserved cell line No. 45 are as follows: (1) GGTA1 gene sequence alignment.

[0077] Wild-type cell line sequence (SEQ ID NO:45): ATGAATGTCAAAGGAAGAGTGGTTCTGTCAATGCTGCTTGTCTCAACTGTAATGTGTTGTGTTTTGGGAATACATCAACAG.

[0078] Cell line sequence 45 (4 bases knocked out on both chromosomes) (SEQ ID NO:46): TATTTGTTGAAAGAAAGATTGTGTCTGT…(4bp)…GCTGGTTGCCTCAACTGAATGGAATGGGTGTTGTTAATACATCCACAG.

[0079] (2) CMAH gene sequence alignment.

[0080] Wild-type cell line sequence (SEQ ID NO:47): ATCACGTACCTTACTCACGCCTGCATGGACCTCAAGCTGGGAGACAAGAGGATGGTGTTCGATCCTTGGTTAATCGGTCCTGCTTTTGCGCGAGGATGGTGGTTACTACACGAGCCTCCATCTGATTGGCTGGAGAGCTGAGCCGCGCAGATTTAATTTACATCAGTCACATGCACTCAGACCACCTGAG.

[0081] Cell line sequence 45 (two bases knocked out on both chromosomes) (SEQ ID NO:48): ATCACGTACCTTACTCACGCCTGCATGGACCTCAAGCTGGGAGACAAGAGGATGGTGCTCGACCCTTGGTTAATCGGTCCTGCTTTTGCGCGAGGATGGTGGTTACTACACGAGCCTCCATCTGATTGGCTGGAGAGCTGAGCCGCGCAGATTTAATTTACATCAGTCACATGC…(2bp)…TCAGACCACCTGAG.

[0082] (3) Alignment of the β4GALNT2 gene sequence.

[0083] Wild-type cell line sequence (SEQ ID NO:49): AGAAGGGCTCCCTCGCCCACCGCCCCTGCTGGCTCAGCCCAACCTCCCCTTTGGGTACCCGGTCCACGGGGTGGAAGTGATGCCTCTACACACCATCCCCATCCCAG.

[0084] Cell line sequence 45 (one base knocked out on each of the two chromosomes) (SEQ ID NO:50): AGAAGGGCTCCCTCGCCCACCGCCCCTGCTGGCTCAGCCCAACCTCCCCTTTGGGTACCCGGTCCACGGG…(1bp)…TGGAAGTGATGCCTCTACACACCATCCCCATCCCA.

[0085] Existing xenotransplant pigs are mainly prepared in steps, and gene overexpression is mainly carried out using the transposon system. This strategy has the following problems: (1) The preparation time is long and multiple cloning is required to obtain multi-gene edited pigs; (2) The integration site of the transposon system is uncertain, and it is impossible to control the copy number and expression level of the inserted gene, which is not conducive to the breeding and stability of gene-edited pig breeds.

[0086] Addressing the industry pain points of existing research, which often focuses on single-gene modification, involves lengthy and complex construction processes, and suffers from insufficient genetic stability and reproducibility of models, this invention utilizes optimized multi-gene editing technology and systems to achieve efficient integration and stable expression of the deletion of three major antigens (GGTA1, CMAH, β4GALNT2) and seven human regulatory factors (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, EPCR), resulting in the production of multi-gene-edited cell line No. 45. This No. 45 cell line was used to prepare multi-gene-edited pigs, which were then used as donor pigs for xenotransplantation. This significantly reduces the recipient's immune rejection of xenotransplanted organs, greatly improving the survival time and physiological function continuity of xenotransplanted organs in the recipient body. This provides a standardized and systematic key technological pathway for the translation of xenotransplantation from basic research to clinical application.

Claims

1. A multi-gene editing system suitable for xenotransplantation, wherein, The multi-gene editing system includes a recombinant DNA sequence capable of simultaneously knocking out three genes, GGTA1, CMAH, and β4GALNT2, and site-specific integration of four genes, hCD46, hCD55, hCD39, and hCD47; and a recombinant DNA sequence capable of site-specific integration of three genes, hCD59, hTBM, and EPCR.

2. The multi-gene editing system according to claim 1, wherein, The recombinant DNA fragment sequences are shown in SEQ ID NO:41 and SEQ ID NO:

43.

3. The multi-gene editing system according to claim 2, wherein, The multi-gene editing system includes expression vector H11 pUC57 containing the DNA fragment shown in SEQ ID NO:41 and expression vector Rosa26 PUC57 containing the DNA fragment shown in SEQ ID NO:

43. The sequence of expression vector H11 pUC57 is shown in SEQ ID NO:42; the sequence of expression vector Rosa26 PUC57 is shown in SEQ ID NO:

44.

4. The use of the multi-gene editing system according to any one of claims 1-3 in the preparation of multi-gene edited cell lines suitable for xenotransplantation, or in the preparation of multi-gene edited animals suitable for xenotransplantation, or in xenotransplantation.

5. Multi-gene edited cell lines or multi-gene edited pigs prepared using the multi-gene editing system according to any one of claims 1-3.

6. The application of the multi-gene edited cell line or multi-gene edited pig as described in claim 5 in xenotransplantation.

7. A multi-gene-edited cell line, wherein, The cell line is Sus scrofa YZ10, with accession number GDMCCNo: 68094.

8. A method for preparing a multi-gene-edited cell line, wherein, The method includes the following steps: S1: Synthesize the sequences shown in SEQ ID NO:41 and SEQ ID NO:43, and then clone the synthesized sequences into the pUC57 vector to obtain expression vector H11 pUC57 and expression vector Rosa26 PUC57; or directly synthesize the expression vector H11 pUC57 and expression vector Rosa26 PUC57 as described in claim 3. S2: Pig cells were transfected with expression vectors H11 pUC57 and Rosa26 pUC57, and then positive cell lines were obtained by screening that simultaneously knocked out three genes (GGTA1, CMAH, β4GALNT2) and integrated seven genes (hCD46, hCD55, hCD39, hCD47, hCD59, hTBM, and EPCR).

9. The multi-gene edited cell line prepared by the preparation method according to claim 8.

10. The use of the cell line of claim 7 or 9 in the preparation of multi-gene-edited pigs or in the preparation of multi-gene-edited pigs for xenotransplantation.

Images