WO2019154437A1 - CRISPR/Cas9载体组合及其在基因敲除中的应用 - Google Patents

CRISPR/Cas9载体组合及其在基因敲除中的应用 Download PDF

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WO2019154437A1
WO2019154437A1 PCT/CN2019/078060 CN2019078060W WO2019154437A1 WO 2019154437 A1 WO2019154437 A1 WO 2019154437A1 CN 2019078060 W CN2019078060 W CN 2019078060W WO 2019154437 A1 WO2019154437 A1 WO 2019154437A1
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crispr
gene
cas9 vector
ggta1
β4galnt2
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PCT/CN2019/078060
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戴一凡
杨海元
王盈
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南京医科大学
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Definitions

  • the invention belongs to the technical field of genetic engineering, and particularly relates to a CRISPR/Cas9 vector and its application in gene knockout.
  • GBHV glutaraldehyde-fixed wild-type heart valve from pig or bovine tissue
  • GGTA1 ⁇ -1,3-galactosyltransferase
  • CMAH CMP-N-acetylneuraminic hydroxylase
  • ⁇ 4GalNT2 ⁇ -1,4-N-acetylgalactosamine
  • the present invention provides a GGTA1/CMAH/ ⁇ 4GalNT2-CRISPR/Cas9 vector, and a further object of the present invention is to provide GGTA1/CMAH/ ⁇ 4GalNT2- The use of the CRISPR/Cas9 vector in knockout of the GGTA1/CMAH/ ⁇ 4GalNT2 gene.
  • the SgRNA combination of the present invention comprises a SgRNA that specifically targets the GGTA1 gene, an SgRNA that specifically targets the CMAH gene, and an SgRNA that specifically targets the ⁇ 4GalNT2 gene; the SgRNA nucleoside that specifically targets the GGTA1 gene
  • the acid sequence is shown in SEQ ID No: 1
  • the SgRNA nucleotide sequence specifically targeting the CMAH gene is shown in SEQ ID No: 2
  • the SgRNA nucleotide sequence specifically targeting the ⁇ 4GalNT2 gene is SEQ. ID No: 3 is shown.
  • a further object of the invention is to provide a CRISPR/Cas9 vector combination comprising a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a ⁇ 4GalNT2-CRISPR/Cas9 vector; the GGTA1-CRISPR/Cas9 vector comprising SEQ ID No:1
  • the ⁇ 4GalNT2-CRISPR/Cas9 vector comprises the nucleotide sequence shown in SEQ ID No: .
  • the nucleotide sequence of the GGTA1-CRISPR/Cas9 vector is shown in SEQ ID No: 4; the nucleotide sequence of the CMAH-CRISPR/Cas9 vector is shown in SEQ ID No: 5; the ⁇ 4GalNT2-CRISPR/ The nucleotide sequence of the Cas9 vector is shown in SEQ ID No: 6.
  • the CRISPR/Cas9 vector was prepared as follows:
  • the SgRNA nucleotide sequence in the step (2) is as shown in SEQ ID No: 1; when the CRISPR/Cas9 vector is CMAH-CRISPR/Cas9 In the step (2), the SgRNA nucleotide sequence is as shown in SEQ ID No: 2; when the CRISPR/Cas9 vector is a ⁇ 4GalNT2-CRISPR/Cas9 vector, the SgRNA nucleotide in the step (2) The sequence is shown in SEQ ID No: 3.
  • a still further object of the present invention is to provide an application of the CRISPR/Cas9 vector combination for knocking out a gene comprising a GGTA1 gene, a CMAH gene and a ⁇ 4GalNT2 gene; comprising the steps of:
  • step (1) The fibroblasts obtained in step (1) were screened for resistance using G418 antibiotics, and the resistant fibroblasts were subjected to PCR amplification gene sequencing to obtain the fibroblasts of knockout GGTA1 gene, CMAH gene and ⁇ 4GalNT2 gene. cell.
  • a still further object of the present invention is to provide the use of the CRISPR/Cas9 vector combination for the preparation of a porcine heart valve that knocks out the GGTA1 gene, the CMAH gene and the ⁇ 4GalNT2 gene;
  • step (1) The fibroblasts obtained in step (1) were screened for resistance using G418 antibiotics, and the resistant fibroblasts were subjected to PCR amplification gene sequencing to obtain the fibroblasts of knockout GGTA1 gene, CMAH gene and ⁇ 4GalNT2 gene. cell;
  • the genome of the pig produced in the step (4) is extracted, amplified by PCR primers, and genotyped.
  • a still further object of the present invention is to provide use of the SgRNA combination in the preparation of a GGTA1 gene, a CMAH gene and a ⁇ 4GalNT2 gene knockout kit.
  • the knockout method is a frameshift mutation, and the above three genes can be completely knocked out to obtain three genes. Knock out the pig and obtain its heart valve;
  • Figure 1 is a schematic diagram of the CRISPR/Cas9 target of the GGTA1, CMAH and ⁇ 4GalNT2 genes;
  • Figure 2 is a schematic diagram of the GGTA1-CRISPR/Cas9 vector
  • Figure 3 is a schematic diagram of the CMAH-CRISPR/Cas9 carrier
  • Figure 4 is a schematic diagram of the ⁇ 4GalNT2-CRISPR/Cas9 vector
  • Figure 5 is a photograph (A) and genotypic identification results (B) of the three-knockout piglets born after somatic cell nuclear transfer;
  • Figure 6 shows the specific binding of ⁇ -1,3-galactosyltransferase (GGTA1), CMP-N-acetylneuraminic hydroxylase (CMAH) and ⁇ -1,4-N-acetylgalactosamine transfer Antibody detection of enzyme 2 ( ⁇ 4GalNT2) detects knockdown of antigen expression in porcine PBMC;
  • GGTA1 ⁇ -1,3-galactosyltransferase
  • CMAH CMP-N-acetylneuraminic hydroxylase
  • ⁇ 4GalNT2 ⁇ -1,4-N-acetylgalactosamine transfer
  • Figure 7 shows GGTA1/CMAH/ ⁇ 4GalNT2 triple knockout pig (TKO), wild type pig (WT), GGTA1 knockout pig (GGTA1-KO) and human isolated PBMC, respectively. After incubation with human serum for 2 hours, PBMC was used. Anti-IgG and IgM antibody binding, flow cytometry to detect binding to human immunoglobulin;
  • Figure 8 is a stress-strain diagram of GGTA1/CMAH/ ⁇ 4GalNT2 three-knockout pig (TKO) and wild-type pig (WT), and the left panel is a stress map of TKO and WT pig heart valves, showing no significant difference between the two; The graph on the right shows the strain corresponding to the stress of the TKO and WT pig heart valves. The results show no significant difference between the two.
  • sgRNA single guide RNA
  • pX330 was used as the backbone plasmid to construct GGTA1-CRISPR/Cas9 vector and CMAH-CRISPR/Cas9 vector respectively.
  • ⁇ 4GalNT2-CRISPR/Cas9 vector was synthesized, and pX330 was used as the backbone plasmid to construct GGTA1-CRISPR/Cas9 vector and CMAH-CRISPR/Cas9 vector respectively.
  • the GGTA1-CRISPR/Cas9 vector was prepared as follows:
  • the exon3 exon3 of GGTA1 gene was selected as the CRISPR/Cas9 target.
  • the 5' end is G and the 3' end is PAM sequence (NGG).
  • the SgRNA sequence was designed to be GAAAATAATGAATGTCAA, see Figure 1, and the nucleotide sequence is shown in SEQ ID No: 1.
  • the GGTA1-CRISPR/Cas9 vector was prepared as follows:
  • Step 1 According to the design principle of cas9 target: the 5' end is G, and the 3' end is PAM sequence (NGG), and the target position is found on the GGTA1 gene;
  • Step 2 Purchasing the pX330 backbone plasmid expressing hSpCas9 and gRNA (Addgene plasmid 423230);
  • Step 3 The company synthesizes the 5'-terminal phosphorylated oligonucleotide chain SgRNA sequence GAAAATAATGAATGTCAA.
  • the SgRNA sequence was cloned into the pX330 backbone vector by the following steps:
  • the digested pX330 plasmid run agarose gel (agarose gel concentration of 1%, ie, 1 g agarose gel was added to 100 mL of running buffer), and purified by a gel recovery kit (QIAGEN). ;
  • step 5 (2) adding 15 ⁇ L of the error-attached plasmid solution obtained in step 5 to the centrifuge tube containing the competent cells, mixing and allowing to stand in an ice bath for 30 min;
  • the constructed CRSAPR/Cas9 vector was named GGTA1-PX330, and the whole nucleotide sequence was as shown in SEQ ID No: 4.
  • CMAH-CRISPR/Cas9 vector was prepared as follows:
  • exon6 of exon 6 of CMAH gene was selected as the CRISPR/Cas9 target.
  • the 5' end is G and the 3' end is PAM sequence (NGG).
  • the SgRNA guide sequence was designed to be GAGTAAGGTACGTGATCTGT, see Figure 1, and the nucleotide sequence SEQ ID No: 2.
  • CMAH-CRISPR/Cas9 vector was prepared as follows:
  • Step 1 According to the design principle of cas9 target: the 5' end is G, and the 3' end is PAM sequence (NGG), and the target position is found on the CMAH gene;
  • Step 2 Purchasing the pX330 backbone plasmid expressing hSpCas9 and gRNA (Addgene plasmid 423230);
  • Step 3 The company synthesizes the 5'-terminal phosphorylated oligonucleotide chain SgRNA sequence GAGTAAGGTACGTGATCTGT.
  • the SgRNA sequence was cloned into the pX330 backbone vector by the following steps:
  • the digested pX330 plasmid run agarose gel (agarose gel concentration of 1%, ie, 1 g agarose gel was added to 100 mL of running buffer), and purified by a gel recovery kit (QIAGEN). ;
  • step 5 (2) adding 15 ⁇ L of the error-attached plasmid solution obtained in step 5 to the centrifuge tube containing the competent cells, mixing and allowing to stand in an ice bath for 30 min;
  • the constructed CRSAPR/Cas9 vector was named CMAH-PX330, and the whole nucleotide sequence is shown as SEQ ID No: 5.
  • the ⁇ 4GalNT2-CRISPR/Cas9 vector was prepared as follows:
  • the exon8 exon8 of ⁇ 4GalNT2 gene was selected as the CRISPR/Cas9 target.
  • the 5′ end is G and the 3′ end is PAM sequence (NGG).
  • the design guide sequence GGTAGTACTCACGAACACTC is shown in Figure 1, and the nucleotide sequence is shown in SEQ ID No: 3.
  • the ⁇ 4GalNT2-CRISPR/Cas9 vector was prepared as follows:
  • Step 1 According to the design principle of cas9 target: the 5' end is G, the 3' end is PAM sequence (NGG), and the target position is found on the ⁇ 4GalNT2 gene;
  • Step 2 Purchasing the pX330 backbone plasmid expressing hSpCas9 and gRNA (Addgene plasmid 423230);
  • Step 3 The company synthesizes a 5'-terminal phosphorylated oligonucleotide chain SgRNA sequence GGTAGTACTCACGAACACTC.
  • the SgRNA sequence was cloned into the pX330 backbone vector by the following steps:
  • the digested pX330 plasmid run agarose gel (agarose gel concentration of 1%, ie, 1 g agarose gel was added to 100 mL of running buffer), and purified by a gel recovery kit (QIAGEN). ;
  • step 5 (2) adding 15 ⁇ L of the error-attached plasmid solution obtained in step 5 to the centrifuge tube containing the competent cells, mixing and allowing to stand in an ice bath for 30 min;
  • the constructed CRSAPR/Cas9 vector was named ⁇ 4GalNT2-PX330, and the entire nucleotide sequence is shown as SEQ ID No: 6.
  • This transgenic fragment (see Figures 2, 3 and 4), which expresses the GGTA1/CMAH/ ⁇ 4GalNT2 gene in mammals, respectively, contains the U6 promoter, and CMV combined with chicken ⁇ -actin (CMV-chicken- ⁇ -actin enhancer)
  • CMV-chicken- ⁇ -actin enhancer chicken ⁇ -actin
  • An enhancer of a gene which has a resistance gene for screening in mammalian cells, a neomycin gene, and an ampicillin gene for screening in a prokaryotic cell.
  • the U6 promoter of the widely expressed ⁇ -skeletal muscle actin (CMV-chicken- ⁇ -actin promoter) gene ensures broad expression of downstream genes.
  • the constructed GGTA1-CRISPR/Cas9 vector, CMAH-CRISPR/Cas9 vector and ⁇ 4GalNT2-CRISPR/Cas9 vector were co-transfected into porcine fetal fibroblasts with the tdTomato plasmid.
  • Single cell clones were obtained by G418 screening, GGTA1/CMAH/ ⁇ 4GalNT2 triple knockout porcine fetal fibroblasts were obtained by sequencing, and GGTA1/CMAH/ ⁇ 4GalNT2 three gene knockout Landrace pigs were prepared by somatic cell nuclear transfer (SCNT).
  • SCNT somatic cell nuclear transfer
  • Step 1 Porcine primary fibroblast recovery
  • the cell complete medium was formulated as: 16% fetal bovine serum (Gibco) + 84% DMEM medium (Gibco), 16% and 84% by volume.
  • Step 2 Co-transfection of porcine primary fibroblasts with constructed GGTA1-PX330, CMAH-PX330, ⁇ 4GalNT2-PX330 and tdTomato plasmids (Clontech, PT4069-5)
  • the three plasmids and the Tdtomato plasmid were added to the 100 ⁇ L nuclear transfer reaction solution obtained in the first step at a mass ratio of 5:1, and mixed, and no bubbles were generated during the process;
  • the cell suspension prepared in the first step was washed twice with DPBS Dulbecic Acid Buffer (Gibco), digested at 37 ° C for 2 min, and the digestion was terminated with DMEM complete medium containing 10% fetal bovine serum, and centrifuged at 1500 rpm. 5 min, discard the supernatant, resuspend the cells using the nuclear transfer reaction solution containing the plasmid in step 2, and avoid the generation of bubbles during the resuspension;
  • DPBS Dulbecic Acid Buffer Gibco
  • the cell culture medium was changed to a complete medium containing 1 mg/mL G418, and the cells were cultured in a constant temperature incubator at 37 ° C, 5% CO 2 , and replaced every 2 to 3 days.
  • the drug concentration of G418 was gradually decreased according to the cell growth condition, and the final concentration of G418 was 0.3 mg/mL, and the G418-resistant monoclonal cell line was gradually grown in the culture dish for 10 to 14 days;
  • the cell line was picked using a cloning ring, and the picked monoclonal cell line was inoculated into a 24-well plate plated with 0.3 mg/mL G418 complete medium, and placed in a constant temperature incubator at 37 ° C, 5% CO 2 . The culture is carried out, and the cell culture medium is changed every 2 to 3 days;
  • the cells in the wells of the 24-well plate are filled with the bottom of the well, and the cells are digested with trypsin and collected, and 4/5 of the cells are inoculated into a 12-well plate or a 6-well plate containing 0.3 mg/mL of G418 complete medium (according to Cell volume), the remaining 1/5 of the cells were left in a 24-well plate to continue the culture;
  • PCR primer sequences are:
  • the forward primer is: 5'-CCTTAGTATCCTTCCCAACCCAGAC-3'
  • the reverse primer is: 5'-GCTTTCTTTACGGTGTCAGTGAATCC-3'
  • the target product of PCR is 428 bp in length
  • the forward primer is: 5'-CTTGGAGGTGATTTGAGTTGGG-3'
  • the reverse primer is: 5'-CATTTTCTTCGGAGTTGAGGGC-3'
  • the length of the PCR target product is 485 bp
  • the forward primer is: 5'-CCCAAGGATCCTGCTGCC-3'
  • the reverse primer is: 5'-CGCCGTGTAAAGAAACCTCC-3';
  • the target product of PCR is 399 bp in length
  • the GGTA1/CMAH/ ⁇ 4GalNT2 target gene was amplified by PCR reaction.
  • the PCR reaction system was as follows:
  • Amplification of the CMAH target gene is the same as the above procedure; amplification of the ⁇ 4GalNT2 target gene is the same as the above procedure.
  • the PCR reaction product was subjected to agarose gel electrophoresis (1%, ie, 1 g agarose gel was added to 100 mL of running buffer). After electrophoresis, the target band was cut under ultraviolet light, and then the gel recovery kit (QIAGEN) was used. Recycling the band of interest and determining the concentration of the recovered PCR product using NanoDrop 200;
  • the PCR product was recovered using TAKARA pMD TM 18-T Vector Cloning Kit links T vector, vector T reaction system as follows:
  • reaction condition of the T vector linkage is 16 ° C reaction for 30 min;
  • the T vector-linked product obtained in the step 5 is transformed with competent cells (TIANGEN), and after transformation, the competent cells are coated on Amp-resistant LB agar solid medium, and cultured in a 37 ° C constant temperature incubator overnight;
  • the knockout efficiencies of the GGTA1, CMAH, and ⁇ 4GalNT2 genes were 56%, 63%, and 41%, respectively.
  • the GGTA1/CMAH/ ⁇ 4GalNT2 tri-knock knockout is significantly reduced in binding to human IgM and IgG, so a three-gene knockout is necessary.
  • the mature oocyte in the step (1) is enucleated by a microscopic operating system, and then the GGTA1/CMAH/ ⁇ 4GalNT2 knockout monoclonal cell line obtained in the fourth step is recovered, and the GGTA1/CMAH/ ⁇ 4GalNT2 knockout cell is used as the knockout cell.
  • the nuclear donor is injected into the enucleated oocyte, and each enucleated oocyte is injected with a GGTA1/CMAH/ ⁇ 4GalNT2 knockout cell;
  • the injected cells were activated by nuclear fusion using electrofusion technology, and the embryos were cultured in a 38.5 ° C incubator for 5 days to develop into mulberry embryos;
  • the GGTA1/CMAH/ ⁇ 4GalNT2 gene knockout piglet is cut out of the ear tissue of the piglet after birth, and then the piglet genomic DNA is extracted using the blood/cell/tissue genomic DNA extraction kit (TIANGEN);
  • PCR reaction was carried out using the pig genomic DNA obtained in the first step, and the PCR reaction conditions were the same as those in the step 4, and then the PCR reaction product was sent to the sequencing company for sequencing, and the sequencing result was compared with the GGTA1/CMAH/ ⁇ 4GalNT2 gene target sequence. Correct.
  • Valve extraction process After instilling, remove the heart from the pig, peel off the envelope outside the heart, and then wash it with PBS, gently peel off the adipose tissue outside the pericardium, and then wash it twice with PBS, then use 0.2% glutaric The aldehyde was fixed for at least 48 h for subsequent experiments.
  • peripheral blood mononuclear cells PBMC
  • IgM, IgG immunoglobulin
  • Example 3 prepared ⁇ -knockout piglet ⁇ -1,3-galactosyltransferase (GGTA1), CMP-N-acetylneuraminic hydroxylase (CMAH) and ⁇ -1,4-N - acetylgalactosyltransferase 2 ( ⁇ 4GalNT2) three antigens were successfully knocked out, as shown in Figure 6, where PBS Control was a blank control, Isotype Control was chicken IgY, WT was wild-type pig, and GGTA1-KO was GGTA1 gene.
  • GGTA1 ⁇ -1,3-galactosyltransferase
  • CMAH CMP-N-acetylneuraminic hydroxylase
  • ⁇ 4GalNT2 ⁇ -1,4-N - acetylgalactosyltransferase 2
  • CMAH-KO was a CMAH knockout pig
  • ⁇ 4GalNT2-KO was a ⁇ 4GalNT2 knockout pig.
  • the results showed that GGTA1/CMAH/ ⁇ 4GalNT2 knockout pigs did not express these three antigens, indicating that the three genes were successfully knocked out.
  • PBMC peripheral blood cell lysate
  • BD red blood cell lysate
  • PBMC precipitate was obtained by pre-cooling a washing solution of 0.1% FBS (solvent as PBS, 0.1%, i.e., 0.1 g of FBS/100 mL of PBS) (enhanced cell sedimentation), rinsing, and centrifugation.
  • PBMC Peripheral blood mononuclear cells
  • the obtained PBMC was incubated, incubated on ice for 2 h, centrifuged at 5000 rpm for 5 min, washed three times with PBS, and the volume ratio was 10% goat serum at 4 ° C for 30 min, then washed with PBS. three times. After incubating the human-specific immunoglobulin antibody, the antibody was washed off with PBS, resuspended, and the average fluorescence intensity was measured on the machine.
  • the pericardium of fresh wild-type and three-knockout pigs were taken and fixed with glutaraldehyde for more than 48 hours.
  • the pericardium was cut into a dumbbell shape of 14 mm long, 2 mm wide, and 2 mm thick.
  • Six samples per group 14.67 ⁇ 1.03 mm long, 2.15 ⁇ 0.23 mm wide, 0.2 ⁇ 0.01 mm thick.
  • the stress and strain of the pericardium were measured using an instron 5943 single column tensile tester. The results showed that there was no significant difference in the mechanical properties of the pericardium of the three knockout pigs and the wild type, as shown in Fig. 7 and Fig. 8.

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Abstract

提供了SgRNA组合，包括特异性靶向GGTA1基因的SgRNA、特异性靶向CMAH基因的SgRNA和特异性靶向β4GalNT2基因的SgRNA；还提供了CRISPR/Cas9载体组合，包括GGTA1-CRISPR/Cas9载体、CMAH-CRISPR/Cas9载体和β4GalNT2-CRISPR/Cas9载体；还提供了CRISPR/Cas9载体组合在敲除GGTA1基因、CMAH基因和β4GalNT2基因中的应用。通过设计特异性靶向的SgRNA序列，三个基因敲除效率分别为56％、63％和41％，敲除与免疫排斥反应有关的三个基因，得到三基因敲除猪，并取得其心脏瓣膜。

Description

CRISPR/Cas9载体组合及其在基因敲除中的应用 技术领域
本发明属于基因工程技术领域，具体涉及一种CRISPR/Cas9载体及其在基因敲除中的应用。
背景技术
目前临床上用于心脏替代治疗的是机械人工心脏瓣膜或者戊二醛固定的来自于猪或者牛组织的野生型心脏瓣膜，简称GBHV。虽然已经应用于临床，但是存在一些问题导致移植效果不佳。首先，戊二醛固定会使血管内皮细胞失去活性，破坏移植效果；其次，GBHV的钙化会导致结构性瓣膜退化，不得不再次手术，从而使发病率和死亡率上升；再次，有研究表明，在65岁以上的人群中，GBHV移植成功10年以上占90％，而在青少年中，移植成功3年的仅为18％，引起不同年龄移植结果显著差异的原因是年轻人损耗快，免疫系统更强大，排斥反应更明显。而引起钙化和结构性瓣膜退化的主要因素就是抗体介导的免疫排斥反应。
猪器官和组织中大量表达的α-1,3-半乳糖基转移酶(GGTA1)，CMP-N-乙酰神经氨酸羟化酶(CMAH)和β-1,4-N-乙酰氨基半乳糖转移酶2(β4GalNT2)是三种引起异种移植免疫排斥的主要抗原(Estrada,J.L.,et al.,Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/beta4GalNT2 genes.Xenotransplantation,2015.22(3):p.194-202)。
发明内容
发明目的：为了解决现有心脏替代治疗出现的抗体介导的免疫排斥反应，本发明提供了一种GGTA1/CMAH/β4GalNT2-CRISPR/Cas9载体，本发明进一步的目的是提供GGTA1/CMAH/β4GalNT2-CRISPR/Cas9载体在敲除GGTA1/CMAH/β4GalNT2基因中的应用。
技术方案：本发明所述SgRNA组合，包括特异性靶向GGTA1基因的SgRNA、特异性靶向CMAH基因的SgRNA和特异性靶向β4GalNT2基因的SgRNA；所述特异性靶向GGTA1基因的SgRNA核苷酸序列如SEQ ID No:1所示，所述特异性靶向CMAH基因的SgRNA核苷酸序列如SEQ ID No:2所示，所述特异性靶向β4GalNT2基因的SgRNA核苷酸序列如SEQ ID No:3所示。
本发明进一步的目的是提供CRISPR/Cas9载体组合，包括GGTA1-CRISPR/Cas9载体、CMAH-CRISPR/Cas9载体和β4GalNT2-CRISPR/Cas9载体；所述GGTA1-CRISPR/Cas9载体含有SEQ ID No:1所示的核苷酸序列，所述CMAH-CRISPR/Cas9载体含有SEQ ID No:2所示的核苷酸序列，所述β4GalNT2-CRISPR/Cas9载体含SEQ ID No:3所示的核苷酸序列。
所述GGTA1-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:4所示；所述CMAH-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:5所示；所述β4GalNT2-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:6所示。
所述CRISPR/Cas9载体按如下方法制备得到：
(1)用限制性内切酶消化pX330质粒，酶切后的质粒使用琼脂糖凝胶分离，用胶回收试剂盒纯化回收酶切产物；
(2)将SgRNA序列按如下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃；
(3)将步骤(1)得到的酶切产物和步骤(2)退火后的SgRNA序列使用连接酶进行连接；
(4)用质粒安全核酸外切酶处理步骤(3)得到的体系，去除错误连接的质粒；
(5)将质粒转化到感受态细胞中进行培养；
(6)从步骤(5)培养的感受态细胞中提取质粒进行测序，确定载体构建成功；
当所述CRISPR/Cas9载体为GGTA1-CRISPR/Cas9载体时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:1所示；当所述CRISPR/Cas9载体为CMAH-CRISPR/Cas9时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:2所示；当所述CRISPR/Cas9载体为β4GalNT2-CRISPR/Cas9载体时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:3所示。
本发明更进一步的目的是提供所述CRISPR/Cas9载体组合在敲除含有GGTA1基因、CMAH基因和β4GalNT2基因中的应用；包括以下步骤：
(1)将CRISPR/Cas9载体组合转化至猪的胎儿成纤维细胞中；
(2)使用G418抗生素对步骤(1)得到的成纤维细胞进行抗性筛选，将具有抗性的成纤维细胞进行PCR扩增基因测序，获得敲除GGTA1基因、CMAH基因和β4GalNT2基因的成纤维细胞。
本发明更进一步的目的是提供所述CRISPR/Cas9载体组合在制备敲除GGTA1基因、CMAH基因和β4GalNT2基因的猪心脏瓣膜中的应用；包括以下步骤：
(1)将CRISPR/Cas9载体组合转化至猪的胎儿成纤维细胞中；
(2)使用G418抗生素对步骤(1)得到的成纤维细胞进行抗性筛选，将具有抗性的成纤维细胞进行PCR扩增基因测序，获得敲除GGTA1基因、CMAH基因和β4GalNT2基因的成纤维细胞；
(3)将步骤(2)得到的成纤维细胞的细胞核移植到去核的猪卵母细胞中培养至囊胚阶段；
(4)将步骤(3)得到的囊胚移植到代孕猪中进行饲养，生产；
(5)提取步骤(4)生产的猪的基因组，利用PCR引物进行扩增，进行基因型鉴定。
本发明更进一步的目的是提供所述SgRNA组合在制备GGTA1基因、CMAH基因和β4GalNT2基因敲除试剂盒中的应用。
有益效果：(1)通过设计特异性靶向GGTA1基因、CMAH基因和靶向β4GalNT2基因的SgRNA序列，三基因敲除效率分别为56％、63％和41％；
(2)通过对猪的基因进行改造，敲除与免疫排斥反应有关的三个基因(GGTA1/CMAH/β4GalNT2)，敲除方式为移码突变，可彻底敲除上述三个基因，得到三基因敲除猪，并取得其心脏瓣膜；
(3)三基因敲除猪的心脏瓣膜与人血清中免疫球蛋白结合显著降低，对克服超急性免疫排斥反应有显著作用，有效解决了异种移植器官短缺和钙化以及结构性瓣膜退化的问题，成为制备GBHV新材料的来源，为异种移植器官供体的基因修饰奠定基础，为临床心脏替代治疗提供宝贵的材料来源。
附图说明
图1为GGTA1、CMAH和β4GalNT2三基因的CRISPR/Cas9靶点示意图；
图2为GGTA1-CRISPR/Cas9载体示意图；
图3为CMAH-CRISPR/Cas9载体示意图；
图4为β4GalNT2-CRISPR/Cas9载体示意图；
图5为体细胞核移植后出生的三基因敲除小猪出生时和断奶后的照片(A)以及基因型鉴定结果(B)；
图6为用特异性结合α-1,3-半乳糖基转移酶(GGTA1)，CMP-N-乙酰神经氨酸羟化酶(CMAH)和β-1,4-N-乙酰氨基半乳糖转移酶2(β4GalNT2)的抗体检测敲除猪PBMC中抗原的表达情况；
图7为GGTA1/CMAH/β4GalNT2三基因敲除猪(TKO)、野生型猪(WT)，GGTA1敲除猪(GGTA1-KO)和人分别分离PBMC，经人的血清孵育PBMC 2小时后，用抗IgG和IgM的抗体结合，流式细胞术检测与人的免疫球蛋白结合情况；
图8为GGTA1/CMAH/β4GalNT2三基因敲除猪(TKO)与野生型猪(WT)的应力应变图，左图为TKO与WT猪心脏瓣膜的应力图，显示两者之间无显著差异；右图为TKO与WT猪心脏瓣膜针对应力所对应的应变，结果显示两者无显著差异。
具体实施方式
实施例1构建CRISPR/Cas9载体
首先根据GGTA1/CMAH/β4GalNT2基因的DNA序列，合成靶向GGTA1，CMAH和β4GalNT2基因的sgRNA(single guide RNA)，以pX330为骨架质粒，分别构建GGTA1-CRISPR/Cas9载体、CMAH-CRISPR/Cas9载体和β4GalNT2-CRISPR/Cas9载体。
一、GGTA1-CRISPR/Cas9载体按如下方法制备得到：
首先根据Genbank中公布的猪GGTA1基因序列，选取GGTA1基因的3号外显子exon3作为CRISPR/Cas9靶点，根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，设计SgRNA序列为GAAAATAATGAATGTCAA，见图1，核苷酸序列如SEQ ID No:1所示。
GGTA1-CRISPR/Cas9载体按如下方法制备得到：
步骤一、根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，在GGTA1基因上寻找靶点位置；
步骤二、购买表达hSpCas9和gRNA的pX330骨架质粒(Addgene plasmid 423230)；
步骤三、公司合成5’端磷酸化寡核苷酸链SgRNA序列GAAAATAATGAATGTCAA。
将SgRNA序列克隆到pX330骨架载体上，具体步骤如下：
1、用限制性内切酶BbsI消化1ug pX330质粒；
2、酶切的pX330质粒跑琼脂糖凝胶(琼脂糖凝胶浓度1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)分离，用胶回收试剂盒(QIAGEN)纯化回收酶切产物；
3、公司合成的5’端磷酸化寡核苷酸链SgRNA序列按照以下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃。
4、按照以下体系启动连接反应：室温反应10min
Figure PCTCN2019078060-appb-000001
5、用质粒安全核酸外切酶处理连接体系，去除错误链接质粒：
Figure PCTCN2019078060-appb-000002
37℃反应30min
6、转化
(1)取50μL感受态细胞(TIANGEN)置于冰浴中；
(2)向装有感受态细胞的离心管中加入15μL步骤5得到的去除错误连接质粒溶液，混匀后在冰浴中静置30min；
(3)将冰浴30min的感受态细胞置于42℃水浴中60～90s，然后迅速转移至冰浴中，使细胞冷却2～3min；
(4)向离心管中加入900μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床150rpm振荡培养45min；
(5)将离心管放到离心机中12000rpm离心5min，然后弃去900μL上清，用剩余的100μL上清重悬感受态细胞沉淀，然后将重悬的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h。
7、小提质粒，公司测序，鉴定打靶质粒构建成功。
所构建的CRSAPR/Cas9载体命名为GGTA1-PX330，全核苷酸序列如SEQ ID No:4 所示。
二、CMAH-CRISPR/Cas9载体按如下方法制备得到：
首先根据Genbank中公布的猪CMAH基因序列，选取CMAH基因的6号外显子exon6作为CRISPR/Cas9靶点，根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，设计SgRNA向导序列为GAGTAAGGTACGTGATCTGT，见图1，核苷酸序列SEQ ID No:2所示。
CMAH-CRISPR/Cas9载体按如下方法制备得到：
步骤一、根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，在CMAH基因上寻找靶点位置；
步骤二、购买表达hSpCas9和gRNA的pX330骨架质粒(Addgene plasmid 423230)；
步骤三、公司合成5’端磷酸化寡核苷酸链SgRNA序列GAGTAAGGTACGTGATCTGT。
将SgRNA序列克隆到pX330骨架载体上，具体步骤如下：
1、用限制性内切酶BbsI消化1ug pX330质粒；
2、酶切的pX330质粒跑琼脂糖凝胶(琼脂糖凝胶浓度1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)分离，用胶回收试剂盒(QIAGEN)纯化回收酶切产物；
3、公司合成的5’端磷酸化寡核苷酸链SgRNA序列按照以下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃。
4、按照以下体系启动连接反应：室温反应10min
Figure PCTCN2019078060-appb-000003
5、用质粒安全核酸外切酶处理连接体系，去除错误链接质粒：
Figure PCTCN2019078060-appb-000004
37℃反应30min
6、转化
(1)取50μL感受态细胞(TIANGEN)置于冰浴中；
(2)向装有感受态细胞的离心管中加入15μL步骤5得到的去除错误连接质粒溶液， 混匀后在冰浴中静置30min；
(3)将冰浴30min的感受态细胞置于42℃水浴中60～90s，然后迅速转移至冰浴中，使细胞冷却2～3min；
(4)向离心管中加入900μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床150rpm振荡培养45min；
(5)将离心管放到离心机中12000rpm离心5min，然后弃去900μL上清，用剩余的100μL上清重悬感受态细胞沉淀，然后将重悬的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h。
7、小提质粒，公司进行测序，鉴定打靶质粒构建成功。
所构建的CRSAPR/Cas9载体命名为CMAH-PX330，全核苷酸序列如SEQ ID No:5所示。
三、β4GalNT2-CRISPR/Cas9载体按如下方法制备得到：
首先根据Genbank中公布的猪β4GalNT2基因序列，选取β4GalNT2基因的8号外显子exon8作为CRISPR/Cas9靶点，根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，设计向导序列GGTAGTACTCACGAACACTC见图1，核苷酸序列SEQ ID No:3所示。
β4GalNT2-CRISPR/Cas9载体按如下方法制备得到：
步骤一、根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，在β4GalNT2基因上寻找靶点位置；
步骤二、购买表达hSpCas9和gRNA的pX330骨架质粒(Addgene plasmid 423230)；
步骤三、公司合成5’端磷酸化寡核苷酸链SgRNA序列GGTAGTACTCACGAACACTC。
将SgRNA序列克隆到pX330骨架载体上，具体步骤如下：
1、用限制性内切酶BbsI消化1ug pX330质粒；
2、酶切的pX330质粒跑琼脂糖凝胶(琼脂糖凝胶浓度1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)分离，用胶回收试剂盒(QIAGEN)纯化回收酶切产物；
3、公司合成的5’端磷酸化寡核苷酸链SgRNA序列按照以下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃。
4、按照以下体系启动连接反应：室温反应10min
Figure PCTCN2019078060-appb-000005
Figure PCTCN2019078060-appb-000006
5、用质粒安全核酸外切酶处理连接体系，去除错误链接质粒：
Figure PCTCN2019078060-appb-000007
37℃反应30min
6、转化
(1)取50μL感受态细胞(TIANGEN)置于冰浴中；
(2)向装有感受态细胞的离心管中加入15μL步骤5得到的去除错误连接质粒溶液，混匀后在冰浴中静置30min；
(3)将冰浴30min的感受态细胞置于42℃水浴中60～90s，然后迅速转移至冰浴中，使细胞冷却2～3min；
(4)向离心管中加入900μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床150rpm振荡培养45min；
(5)将离心管放到离心机中12000rpm离心5min，然后弃去900μL上清，用剩余的100μL上清重悬感受态细胞沉淀，然后将重悬的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h。
7、小提质粒，从公司测序，鉴定打靶质粒构建成功。
所构建的CRSAPR/Cas9载体命名为β4GalNT2-PX330，全核苷酸序列如SEQ ID No:6所示。
这种能广泛存在哺乳动物中分别表达GGTA1/CMAH/β4GalNT2基因的转基因片段(见图2、3和4)包含U6启动子，CMV联合鸡β-肌动蛋白(CMV-chicken-β-actin enhancer)基因的增强子，且带有哺乳动物细胞中筛选用的抗性基因—新霉素(Neomycin)基因和原核细胞中筛选用的抗性基因—氨苄青霉素(ampicillin)基因。这种能广泛性表达的β-骨骼肌肌动蛋白(CMV-chicken-β-actin promoter)基因的U6启动子可保证下游基因广泛性表达。
实施例2利用体细胞克隆的方法构建GGTA1/CMAH/β4GalNT2三基因敲除猪
将构建好的GGTA1-CRISPR/Cas9载体、CMAH-CRISPR/Cas9载体和β4GalNT2-CRISPR/Cas9载体通过与tdTomato质粒共转染猪胎儿成纤维细胞。通过G418筛选获得单细胞克隆，测序鉴定获得GGTA1/CMAH/β4GalNT2三基因敲除的猪胎儿成纤维细胞，通过体细胞核移植(SCNT)制备GGTA1/CMAH/β4GalNT2三基因敲除的长白猪。提取刚出生的小猪基因组，利用PCR引物进行扩增，连接T载体进行基因型鉴定。
步骤一、猪原代成纤维细胞复苏
1、从液氮中取出冻存的原代猪成纤维细胞，在37℃水浴中解冻；
2、将解冻的细胞转入无菌的15mL离心管中，然后加入3mL细胞培养基，1500rpm离心5min；
其中，细胞完全培养基的配方为：16％胎牛血清(Gibco)+84％DMEM培养基(Gibco)，16％和84％为体积百分比。
3、弃去上清，加入2mL完全培养基重悬细胞沉淀，然后将重悬的细胞铺入6cm细胞培养皿中，补加2mL完全培养基，置于37℃，5％CO 2(体积百分比)的恒温培养箱中进行培养；
4、将细胞培养至长满皿底90％左右时使用0.05％(5g/100mL)的胰蛋白酶将细胞消化下来，然后加入完全培养基终止消化，将细胞悬液转入15mL离心管中，1500rpm离心5min，弃去上清，使用2mL完全培养基重悬细胞，对细胞计数，将细胞总量调整至1.5×10 6以备下一步核转染实验。
步骤二、使用构建好的GGTA1-PX330，CMAH-PX330，β4GalNT2-PX330和tdTomato质粒(Clontech,PT4069-5)共转染猪原代成纤维细胞
使用哺乳动物成纤维细胞核转染试剂盒(Lonza)与Lonza Nucleofactor TM2b核转仪进行核转染实验
1、配制核转染反应液，体系如下：
核转染基本溶液     82μL
补充成分           8μL
2、将构建好的三个质粒与Tdtomato质粒分别按照质量比5:1的比例加入本步骤1获得的100μL核转反应液中混匀，过程中注意切勿产生气泡；
3、将步骤一制备得到的细胞悬液使用DPBS杜氏磷酸缓冲液(Gibco)洗两遍，37℃消化2min，用含体积百分比为10％胎牛血清的DMEM完全培养基终止消化后，1500rpm离心5min，弃去上清，使用本步骤2中含有质粒的核转反应液重悬细胞，重悬过程中要避免气泡的产生；
4、将该核转体系小心加入到试剂盒带有的电转杯中，注意防止气泡。先用含有100μLPBS的电转杯放置于Lonza核转仪的杯槽内，选择U023核转程序调试程序后，将含有细胞的电转杯电击转染后立即在超净台内将电转杯中液体轻柔吸出，转入到1mL含体积百分比为16％胎牛血清的DMEM完全培养基中，轻轻混匀；
5、准备含8mL完全培养基的培养皿(10cm)若干，吸取核转后的细胞悬液加入含有完全培养基的培养皿中，混匀，在显微镜下观察细胞数量，计数，使得培养皿在显微镜下一个视野内约有50～60个细胞，其余皿均按照此细胞悬液最终用量加入，混匀后放置于37℃，5％CO 2的恒温培养箱中进行培养。
步骤三、三基因敲除细胞系的筛选
1、将步骤二所得细胞培养24h后将细胞培养基更换为含有1mg/mL G418的完全培养基，放置于37℃，5％CO 2的恒温培养箱中进行培养，每2～3天更换一次细胞培养基，期间根据细胞生长状况逐渐降低G418的药物浓度，G418终浓度为0.3mg/mL，培养10～ 14天左右培养皿中会陆续长出G418抗性的单克隆细胞系；
2、使用克隆环挑取细胞系，将挑取的单克隆细胞系接种于铺有0.3mg/mL G418完全培养基的24孔板中，放置于37℃，5％CO 2的恒温培养箱中进行培养，每2～3天换一次细胞培养基；
3、待24孔板的孔中细胞长满孔底，使用胰蛋白酶消化并收集细胞，其中4/5细胞接种到含有0.3mg/mL G418完全培养基的12孔板或6孔板中(根据细胞量)，剩余的1/5的细胞留在24孔板中继续培养；
4、待12孔板或6孔板细胞铺满孔底后使用0.05％(5g/100mL)的胰蛋白酶消化并收集细胞，使用细胞冻存液(90％胎牛血清+10％DMSO，体积比)将细胞冻存；
步骤四、三基因敲除细胞系的基因鉴定
1、待24孔板中细胞长满孔底后使用0.05％(5g/100mL)的胰蛋白酶消化并收集细胞，然后在细胞中加入25ml NP-40裂解液裂解细胞提取细胞基因组DNA，裂解程序为：55℃60min——95℃5min——4℃，反应结束后基因组DNA于-20℃保存；
2、针对GGTA1/CMAH/β4GalNT2基因靶点信息设计相应的PCR引物，PCR引物序列分别为：
GGTA1
正向引物为：5’-CCTTAGTATCCTTCCCAACCCAGAC-3’
反向引物为：5’-GCTTTCTTTACGGTGTCAGTGAATCC-3’
PCR目的产物长度为428bp；
CMAH
正向引物为：5’-CTTGGAGGTGATTTGAGTTGGG-3’
反向引物为：5’-CATTTTCTTCGGAGTTGAGGGC-3’
PCR目的产物长度为485bp；
β4GalNT2
正向引物为：5’-CCCAAGGATCCTGCTGCC-3’
反向引物为：5’-CGCCGTGTAAAGAAACCTCC-3’；
PCR目的产物长度为399bp；
3、使用PCR反应扩增GGTA1/CMAH/β4GalNT2靶点基因，PCR反应体系如下：
Figure PCTCN2019078060-appb-000008
反应条件如下
Figure PCTCN2019078060-appb-000009
Figure PCTCN2019078060-appb-000010
CMAH靶点基因的扩增同上述步骤；β4GalNT2靶点基因的扩增同上述步骤。
4、将PCR反应产物进行琼脂糖凝胶电泳(1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)，电泳结束后在紫外线下切下目的条带，然后使用胶回收试剂盒(QIAGEN)回收目的条带，并使用NanoDrop 200测定回收的PCR产物的浓度；
5、将回收的PCR产物使用TAKARA pMD TM18-T Vector Cloning Kit链接T载体，T载体反应体系如下：
pMD18-T vector    1μL
胶回收PCR产物     81.7ng*
ddH 2O             补齐体系至10uL
*注：TAKARA pMD TM18-T Vector Cloning Kit说明书上对Insert DNA(本次为胶回收PCR产物)用量的要求0.1～0.3pM，本次选取0.2pM，用量计算方法为：Insert DNA的使用量(ng)＝nmol数×660×Insert DNA的bp数。
T载体链接的反应条件为16℃反应30min；
6、将本步骤5所得的T载体链接产物使用感受态细胞(TIANGEN)进行转化，转化后将感受态细胞涂布于Amp抗性的LB琼脂固体培养基上，37℃恒温培养箱培养过夜；
从培养过夜的培养基上挑取10～15个单克隆菌落送测序公司进行测序，然后将测序结果与靶点GGTA1/CMAH/β4GalNT2信息进行比对，从而判断该细胞系是否为GGTA1/CMAH/β4GalNT2基因敲除细胞系；
本次挑取的单克隆细胞系共27个，其中三个基因同时敲除的双等位基因敲除细胞系1个，编号为50#，该克隆基因型情况见表1：
表1 GGTA1/CMAH/β4GalNT2基因敲除的长白猪成纤维细胞的基因鉴定
Figure PCTCN2019078060-appb-000011
敲除GGTA1、CMAH、β4GalNT2基因的敲除效率分别为56％、63％和41％。
GGTA1/CMAH/β4GalNT2三基因敲除和GGTA1/CMAH两基因敲除相比，与人的IgM，IgG的结合明显降低，因此三基因敲除是必须的。
步骤五、体细胞核移植
1、从屠宰场购买六月龄以上的母猪卵巢，人工抽取卵泡中未成熟的卵母细胞，在显微镜下挑取质量较好的卵母细胞并置于38.5℃，5％CO 2恒温培养箱中培养42～44h至卵母细胞成熟；
2、利用显微操作系统将本步骤(1)中成熟的卵母细胞去核，然后复苏步骤四获得的GGTA1/CMAH/β4GalNT2敲除单克隆细胞系，将GGTA1/CMAH/β4GalNT2敲除细胞作为核供体注入去核卵母细胞中，每个去核卵母细胞注射一个GGTA1/CMAH/β4GalNT2敲除细胞；
3、将注射好的细胞利用电融合技术将核移植后得重构胚胎激活，将胚胎置于38.5℃培养箱培养5天发育成桑椹胚；
4、将发育情况良好的胚胎移植到代孕母猪的子宫中，小心护理代孕母猪，移植一个月后使用B超检测受体猪的怀孕情况，期间及时监控直至代孕母猪分娩。
步骤六、三基因基因敲除巴马小型猪的基因型分析
1、GGTA1/CMAH/β4GalNT2基因敲除小猪出生后剪取小猪耳部组织，然后使用血液/细胞/组织基因组DNA提取试剂盒(TIANGEN)提取小猪基因组DNA；
2、使用本步骤1中所得小猪基因组DNA进行PCR反应，PCR反应条件同步骤四3，然后将PCR反应产物送测序公司进行测序，将测序结果与GGTA1/CMAH/β4GalNT2基因靶点序列进行比对。
本次共出生8只长白公猪编号为1-8，出生的8头公猪与细胞基因型结果一致。
瓣膜提取过程：灌注后杀猪取出心脏，剥离心脏外面的包膜，然后用PBS洗一遍后，轻轻剥离心包膜外面的脂肪组织，再用PBS洗两遍之后，用0.2％的戊二醛固定至少48h，用于后续实验。
实施例3 GGTA1/CMAH/β4GalNT2三基因敲除猪的表型分析
1、敲除野生型猪体内的GGTA1、CMAH和β4GalNT2基因，可以有效地降低异种移植过程中的超急性免疫排斥反应
小猪断奶以后，抽血、分离外周血单个核细胞(PBMC)，通过流式细胞仪测定小猪的基因敲除情况，以及与人血清中免疫球蛋白(IgM，IgG)的结合情况，发现实施例2制得的三基因敲除小猪的α-1,3-半乳糖基转移酶(GGTA1)，CMP-N-乙酰神经氨酸羟化酶(CMAH)和β-1,4-N-乙酰氨基半乳糖转移酶2(β4GalNT2)三种抗原成功被敲除，如图6所示，其中PBS Control为空白对照，Isotype Control为chicken IgY，WT为野生型猪，GGTA1-KO为GGTA1基因敲除猪，CMAH-KO为CMAH基因敲除猪，β4GalNT2-KO为β4GalNT2基因敲除猪，结果显示，GGTA1/CMAH/β4GalNT2敲除猪不表达这三种抗原，代表三基因成功敲除。
通过以下方法分离PBMC：取100μL抗凝血，加3倍体积的红细胞裂解液(BD，用去离子水稀释10倍)，室温裂解5min～10min。离心后，弃上清，用预冷洗涤液0.1％FBS(溶剂为PBS，0.1％即0.1g FBS/100mL PBS)(增强细胞沉降)，漂洗，离心，即获得PBMC沉淀。
2、外周血单个核细胞(PBMC)经流式细胞仪检测三基因敲除小猪和对照野生型小猪的PBMC与人血清中免疫球蛋白结合水平，结果说明与野生型猪相比，三基因敲除猪的PBMC与人的免疫球蛋白结合水平明显降低，接近于人的水平
商业化的人血清56℃水浴锅灭活30min后，孵育已获得的PBMC，冰上孵育2h，5000rpm转离心5min，用PBS洗三次，体积比为10％山羊血清4℃封闭30min后，PBS洗三次。孵育人特异性的免疫球蛋白抗体后，PBS洗掉抗体，重悬，上机检测平均荧光强度。结果发现相较于野生型猪的PBMC来说，三基因敲除猪的PBMC与人的免疫球蛋白结合水平大大降低，与正常情况下人的水平差异不大，如图6所示，GGTA1/CMAH/β4GalNT2三基因敲除猪对克服超急性免疫排斥反应有显著作用。
3、单轴力学测试野生型与三基因敲除猪的心包膜的力学性质。
取新鲜的野生型与三基因敲除猪的心包膜，用戊二醛固定48h以上。将心包膜剪裁成14mm长，2mm宽，2mm厚的哑铃形状。每组6个样品，14.67±1.03mm长，2.15±0.23mm宽，0.2±0.01mm厚。用instron 5943单立柱材料拉伸实验仪检测心包膜的应力和应变。结果显示三基因敲除猪的心包膜与野生型的力学性质无显著差异，如图7、图8所示。
目前临床上应用的戊二醛固定的商业化的心脏瓣膜已经成熟应用于临床，只是由于钙化和免疫排斥反应等原因使心脏瓣膜的作用无法维持很长时间。而敲除了与免疫排斥反应相关的三个基因的猪的心脏瓣膜可以作为一个新型的生物材料瓣膜的来源，为解决临床心脏替代治疗中出现的问题提供解决思路。

Claims (9)

  1. SgRNA组合，其特征在于，包括特异性靶向GGTA1基因的SgRNA、特异性靶向CMAH基因的SgRNA和特异性靶向β4GalNT2基因的SgRNA；其中，所述特异性靶向GGTA1基因的SgRNA核苷酸序列如SEQ ID No:1所示，所述特异性靶向CMAH基因的SgRNA核苷酸序列如SEQ ID No:2所示，所述特异性靶向β4GalNT2基因的SgRNA核苷酸序列如SEQ ID No:3所示。
  2. CRISPR/Cas9载体组合，其特征在于，包括GGTA1-CRISPR/Cas9载体、CMAH-CRISPR/Cas9载体和β4GalNT2-CRISPR/Cas9载体；所述GGTA1-CRISPR/Cas9载体含有SEQ ID No:1所示的核苷酸序列，所述CMAH-CRISPR/Cas9载体含有SEQ ID No:2所示的核苷酸序列，所述β4GalNT2-CRISPR/Cas9载体含SEQ ID No:3所示的核苷酸序列。
  3. 根据权利要求2所述的CRISPR/Cas9载体组合，其特征在于，所述GGTA1-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:4所示；所述CMAH-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:5所示；所述β4GalNT2-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:6所示。
  4. 根据权利要求2所述的CRISPR/Cas9载体组合，其特征在于，所述CRISPR/Cas9载体按如下方法构建得到：
    (1)用限制性内切酶消化pX330质粒，酶切后的质粒使用琼脂糖凝胶分离，用胶回收试剂盒纯化回收酶切产物；
    (2)将SgRNA序列按如下程序退火：
    37℃30min
    95℃5min然后以5℃/min的速率降至25℃；
    (3)将步骤(1)得到的酶切产物和步骤(2)退火后的SgRNA序列使用连接酶进行连接；
    (4)用质粒安全核酸外切酶处理步骤(3)得到的体系，去除错误连接的质粒；
    (5)将质粒转化到感受态细胞中进行培养；
    (6)从步骤(5)培养的感受态细胞中提取质粒进行测序，确定载体构建成功；
    当所述CRISPR/Cas9载体为GGTA1-CRISPR/Cas9载体时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:1所示；当所述CRISPR/Cas9载体为CMAH-CRISPR/Cas9时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:2所示；当所述CRISPR/Cas9载体为β4GalNT2-CRISPR/Cas9载体时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:3所示。
  5. 权利要求2-4任一所述的CRISPR/Cas9载体组合在敲除GGTA1基因、CMAH基因和β4GalNT2基因中的应用。
  6. 根据权利要求5所述的应用，其特征在于，包括以下步骤：
    (1)将CRISPR/Cas9载体组合转化至猪的胎儿成纤维细胞中；
    (2)使用G418抗生素对步骤(1)得到的成纤维细胞进行抗性筛选，将具有抗性的成纤维细胞进行PCR扩增基因测序，获得敲除GGTA1基因、CMAH基因和β4GalNT2基因的成纤维细胞。
  7. 权利要求2-4任一所述的CRISPR/Cas9载体组合在制备敲除了含有GGTA1基因、CMAH基因和β4GalNT2基因的猪心脏瓣膜中的应用。
  8. 根据权利要求7所述的应用，其特征在于，包括以下步骤：
    (1)将CRISPR/Cas9载体组合转化至猪的胎儿成纤维细胞中；
    (2)使用G418抗生素对步骤(1)得到的成纤维细胞进行抗性筛选，将具有抗性的成纤维细胞进行PCR扩增基因测序，获得敲除GGTA1基因、CMAH基因和β4GalNT2基因的成纤维细胞；
    (3)将步骤(2)得到的成纤维细胞的细胞核移植到去核的猪卵母细胞中培养至囊胚阶段；
    (4)将步骤(3)得到的囊胚移植到代孕猪中进行饲养，生产；
    (5)提取步骤(4)生产的猪的基因组，利用PCR引物进行扩增，进行基因型鉴定。
  9. 权利要求1所述SgRNA组合在制备GGTA1基因、CMAH基因和β4GalNT2基因敲除试剂盒中的应用。
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