SgRNA and double gene knockout pig fibroblast edited by using the same

Abstract

This invention provides an sgRNA, the sequence of which is shown in SEQ ID NO.1, SEQ ID NO.4, or SEQ ID NO.7. This invention also provides a method for using the sgRNA to knock out RAG-1 and RAG-2 genes in porcine fibroblasts, as well as a method for RAG-2 and IL2RG, and porcine fibroblasts with the knockout genes. The sgRNA provided by this invention has excellent specificity, cleavage efficiency, and synergistic effects. Using the sgRNA combination provided by this invention, porcine fibroblasts with the knockout genes can be obtained in a single step. These porcine fibroblasts with the knockout genes show promise for use in preparing immunodeficient porcine animal models.

Description

Technical Field

[0001] This invention discloses an sgRNA, a gene editing method, and the obtained gene knockout cells, belonging to the field of nucleic acid and cell engineering. Background Technology

[0002] Pigs are important transgenic large animal models. The development of CRISPR / Cas9 technology has made it possible to rapidly and efficiently improve pig cells. Pigs are similar to humans in body size, lifespan, physiological indicators, and especially immune mechanisms. Pigs with severe comprehensive immunodeficiency (SCID) are important large animal models for medical research. RAG-1 and RAG-2, namely recombination activating genes 1 and 2, are both genes located in the cell nucleus and participate in the DNA recombination process. They play an important role in antibody production and antibody gene differentiation. Mutations in RAG1 or RAG2 genes lead to the complete absence of B cells and T cells. IL-2RG, namely the common γ chain of interleukin-2 receptor (also known as IL-2Rγ, CD132), is a component of multiple cytokine receptors, namely IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, and is therefore called the "common γ chain" (γc). Cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, also known as γ-c cytokines, are the most studied types of cytokines. Mutations in the IL-2RG gene lead to the loss or severe depletion of T cells and natural killer (NK) cells, without affecting the number of B cells. Immunodeficient pigs with the aforementioned gene mutations are important tools in the research of immune and tumor-related diseases. Screening and preparing immunodeficient pigs with the aforementioned gene mutations using traditional mutation methods, especially double-gene mutations, is a time-consuming, tedious, and low-success-rate process.

[0003] The purpose of this invention is to construct a double-gene knockout porcine fibroblast based on CRISPR / Cas9 technology, providing a cellular tool for the preparation of immunodeficient pigs. Summary of the Invention

[0004] To achieve the above objectives, this invention first provides an sgRNA that targets the 5222-5241 base region of the RAG-1 gene (gene sequence information: NC_010444.4:c24573853-24565447 RAG1 [organism=Susscrofa] [GeneID=397506][chromosome=2]) in porcine fibroblasts. In this invention, the sgRNA is designated "RAG1gRNA1", or... The sgRNA targets the 4855-4874 base range of the RAG-2 gene (gene sequence information: NC_010444.4:24547605-24553944 RAG2 [organism=Sus scrofa] [GeneID=100151744][chromosome=2]) in porcine fibroblasts. In this invention, the sgRNA is designated as "RAG2gRNA1", or... The sgRNA targets the 4489th to 5224th base region of the IL2RG gene (gene sequence information: NC_010461.5:c57151242-57143568 IL2RG [organism=Sus scrofa] [GeneID=397156][chromosome=X]) in porcine fibroblasts. In this invention, the sgRNA is designated as "IL2RGgRNA1".

[0005] In a preferred embodiment, the sequence of the sgRNA is as shown in SEQ ID NO.1, or as shown in SEQ ID NO.4, or as shown in SEQ ID NO.7.

[0006] Secondly, the present invention provides a vector containing the above-mentioned sgRNA.

[0007] In a preferred embodiment, the vector is a CRISPR / Cas9 vector.

[0008] In a more preferred embodiment, the vector is LentiGRISPR-v2.

[0009] Third, this invention provides a method for knocking out the RAG-1 gene, RAG-2 gene, and / or IL2RG-2 gene in porcine fibroblasts using the above-mentioned vector, the method comprising the following steps: (1) Transfect the vector containing the above sgRNA into porcine fibroblasts; (2) Pig fibroblasts with gene knockout obtained from the vector through resistance screening of the pig fibroblasts.

[0010] In a preferred embodiment, the vector in step (1) comprises a vector containing sgRNA with the sequence shown in SEQ ID NO.1 and a vector containing sgRNA with the sequence shown in SEQ ID NO.4, or the vector comprises a vector containing sgRNA with the sequence shown in SEQ ID NO.4 and a vector containing sgRNA with the sequence shown in SEQ ID NO.7.

[0011] Fourth, the present invention provides a porcine fibroblast that has undergone double gene knockout, wherein the double gene knockout is a double gene knockout of RAG-1 gene and RAG-2 gene, or a double gene knockout of RAG-2 gene and IL2RG-2 gene.

[0012] In a preferred embodiment, the porcine fibroblasts that have undergone dual gene knockout are obtained by the method described above for knocking out the porcine fibroblast RAG-1 gene, RAG-2 gene, and / or IL2RG-2 gene.

[0013] Finally, the present invention provides the application of the porcine fibroblasts in the preparation of immunodeficient pigs, wherein the application is to transplant the porcine fibroblasts into the maternal pig through somatic cell nuclear transfer.

[0014] The sgRNA cleavage efficiency provided by this invention is greater than 50%. Using the sgRNA provided by this invention, especially the combination of RAG1gRNA1 and RAG2gRNA1, or the combination of RAG2gRNA1 and IL2RGgRNA1, porcine fibroblasts with double gene knockout (i.e., double gene knockout of RAG-1 and RAG-2 genes, or double gene knockout of RAG-2 and IL2RG-2 genes) can be obtained through a single transfection. This demonstrates excellent specificity, synergy, low off-target effects, and low immunogenicity. These double-gene knockout porcine fibroblasts, as a cellular tool, can be transplanted into a somatic cell nuclear transfer model to obtain an immunodeficient pig animal model, showing excellent application prospects in the field of transgenic animal preparation. Attached Figure Description

[0015] Figure 1 .sgRNA in vitro detection results; Figure 2 RAG1-CRISPR-Cas9 sgRNA sequencing results; Figure 3 RAG2-CRISPR-Cas9 sgRNA sequencing results; Figure 4IL2RG-CRISPR-Cas9 sgRNA sequencing results; Figure 5 EcoRI single enzyme digestion electrophoresis results; Figure 6 Sequencing results of RAG2 and RAG1 double gene knockout clones; Figure 7 Sequencing results of RAG2 and IL2RG double gene knockout clones. Detailed Implementation

[0016] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0017] The reagents and instruments used in this invention are as follows: LentiGRISPR-v2 vector (addgene, 52961), porcine ear fibroblasts (isolated and preserved by our research group), the porcine ear fibroblast culture system consisting of DMEM medium (gibco, 11995056) + 10% serum (gibco, A5669701), Guide-it sgRNA In Vitro Transcription Kit for in vitro shearing efficiency assay (TAKARA, catalog number 632635), DNA MicroKit for micro-genomic extraction (QIAGEN, catalog number 56304), EcoRI (NEB, catalog number R3101V); gel extraction kit (Copyright © Suzhou Youyilandi Biotechnology Co., Ltd., catalog number: UE-GX-PLUS-10), Lonza nuclear electroporator (Lonza, HBeco), Lonza electroporation kit (Lonza, V4XP-3024). PCR instrument (MyCycler Thermal Cycler), TA cloning kit (Novazia, C601-02), PCR MIX (Novazia, P525-03).

[0018] Example 1. Screening for sgRNAs with high cleavage activity by in vitro cleavage activity 1.1 Design of sgRNA The efficiency of the CRISPR / Cas9 system mainly depends on the efficiency of sgRNA. Therefore, gene knockout sgRNAs were designed before the experiment. The RAG1 transcript has only one exon, so 3 sgRNA sequences were designed. The RAG2 transcript has only one exon, so 3 sgRNA sequences were designed. The IL2RG gene edits multiple transcripts, so 6 sgRNAs were designed. The 12 sgRNA sequences are shown in Table 1.

[0019] Table 1. sgRNA sequence + NGG .

[0020] 1.2 In vitro synthesis of sgRNA Before conducting research on Cas9-mediated gene editing, the efficiency of different sgRNAs was first tested in vitro. First, a template containing the sgRNA target was generated via PCR. The in vitro primer design had to include three modules: the first segment consisted of a T7 promoter sequence, four bases at the 5' end of the primer, and a transcription start site (GG), totaling 23 bases (as shown in SEQ ID NO. 13). The middle segment contained the specific sgRNA target sequence, totaling 20 bases. The final segment was the annealing sequence guiding the scaffold template (as shown in SEQ ID NO. 14), and the 3' end of the primer contained 15 bases. The primer sequence design is shown in Table 2.

[0021] Table 2. In vitro primer design for PCR amplification of templates containing sgRNA targets .

[0022] 1.3 In vitro transcription of sgRNA 1.3.1 PCR amplification of sgRNA template (1) Mix the following mixture in a PCR tube, briefly vortex and centrifuge.

[0023] Table 3. PCR system ;

[0024] (2) Place the test tube into the PCR instrument and run the following program: (98℃ 10sec, 68℃ 10sec) × 33 cycles, 4℃∞.

[0025] 1.3.2. In vitro transcription (IVT) response (1) The PCR amplification product can be used directly as a template for the IVT reaction without purification. Mix the following mixture in a PCR tube, briefly vortex and centrifuge.

[0026] Table 4. In vitro transcription (IVT) response ;

[0027] (2) Place the test tubes in the PCR instrument and run the following program: 37℃, 4 hr; 4℃, forever;

[0028] (3) After PCR, add 2 µl of recombinant DNase I (RNase-Free) to the system, briefly vortex and centrifuge;

[0029] (4) Place the test tube into the PCR instrument and run the following program: 37℃, 15hr; 4℃, forever.

[0030] 1.4 Purification of transcribed sgRNA Using the IVT RNA Clean-Up Kit, the steps are as follows: 1.4.1 Add 78 µl of RNase-free water to the reaction mixture from the previous step, bringing the total volume to 100 µl. Transfer all the liquid to a 1.5 ml microcentrifuge tube;

[0031] 1.4.2 Add 30 µl of IVT binding buffer and vortex for 5 seconds;

[0032] 1.4.3 Add 130 µl of isopropanol and vortex for 5 seconds;

[0033] 1.4.4 Place the adsorption column in the collection tube and load the sample from step 1.3 onto the column, then centrifuge at 11,000g for 30 seconds at room temperature;

[0034] 1.4.5 Discard the waste liquid at the bottom and return the adsorption column to the collection tube;

[0035] 1.4.6 Add 600 µl of IVT washing buffer and centrifuge at 11,000 g for 30 seconds at room temperature;

[0036] 1.4.7 Discard the waste liquid at the bottom and return the adsorption column to the collection tube;

[0037] 1.4.8 Add 250 µl of IVT washing buffer and centrifuge at 11,000 g for 2 min at room temperature;

[0038] 1.4.9 Transfer the IVT RNA cleaning spin column into a new 1.5 ml centrifuge tube;

[0039] 1.4.10 Add 20 µl of RNase-free water to the column membrane and incubate at room temperature for 1 min;

[0040] 1.4.11 Centrifuge at 11,000g for 1 min at room temperature;

[0041] 1.4.12 The OD value was measured using a spectrophotometer (Nanodrop), and the expected yield of sgRNA was 10-20 µg.

[0042] 1.5 Screening for sgRNAs that effectively cleave their target sites 1.5.1 Extraction of Genomic DNA 1.5.2 PCR amplification of DNA fragments containing sgRNA target sequences (1) Design primers to amplify the target region of CRISPR / Cas9. The optimal amplicon size is 600-800 bp, and the sgRNA target sequence is asymmetrically located within the amplicon; each cleavage fragment is at least 250 bp, and there should be a difference of >100 bp between fragments after Cas9 cleavage. The RAG1F1 / R1 amplification product is located at bases 4897-5712 in the entire genome, and the target sequence of sgRNA1 (SEQ ID NO.1) is located at bases 5222-5241; the RAG1F2 / R2 amplification product is located at bases 5520-6352 in the entire genome, and the target sequence of sgRNA2 (SEQ ID NO.2) is located at bases 5839-5858; the RAG1F3 / R3 amplification product is located at bases 6526-7352 in the entire genome, and the target sequence of sgRNA3 (SEQ ID NO.3) is located at bases 6836-6855.

[0043] The RAG2F1 / R1 amplification product is located at bases 4597-5304 in the entire genome, and the target sequence of sgRNA4 (SEQ ID NO.4) is located at bases 4855-4874; the RAG2F2 / R2 amplification product is located at bases 5048-5821 in the entire genome, and the target sequence of sgRNA5 (SEQ ID NO.5) is located at bases 4854-4873; the RAG2F3 / R3 amplification product is located at bases 5383-6050 in the entire genome, and the target sequence of sgRNA6 (SEQ ID NO.6) is located at bases 5656-5675.

[0044] The IL2RGF1 / R1 amplification product is located at bases 4489-5224 in the entire genome, and the target sequence of sgRNA7 (SEQ ID NO. 7) is located at bases 4763-4782; the IL2RGF2 / R2 amplification product is located at bases 4501-5213 in the entire genome, and the target sequence of sgRNA8 (SEQ ID NO. 8) is located at bases 4790-4809, while the target sequence of sgRNA9 (SEQ ID NO. 9) is located at bases 4908-4927; the IL2RGF3 / R3 amplification product is located at bases 4977-5793 in the entire genome, and the target sequence of sgRNA10 (SEQ ID NO. 10) is located at bases 5244-5263, while the target sequence of sgRNA11 (SEQ ID NO. 10) is located at bases 5244-5263. NO.11) targets sequences from base 5243 to base 5262; the IL2RGF4 / R4 amplification product targets sequences from base 5445 to base 6264 throughout the genome; and the sgRNA12 (SEQ ID NO.12) targets sequences from base 5757 to base 5776. Primer design is as follows: Table 5. PCR primer sequences for sgRNA target sequence .

[0045] (2) Mix the following system in a PCR tube, briefly vortex and centrifuge.

[0046] Table 6. PCR System .

[0047] (3) Place the test tube into the PCR instrument and run the following program: 95℃ for 3 min, (95℃ for 20 sec, 58℃ for 30 sec, 72℃ for 3 min) × 29 cycles, 72℃ for 5 min, 4℃ for ∞.

[0048] (4) The PCR product was excised and recovered to obtain a purified DNA fragment containing the gRNA target sequence.

[0049] 1.5.3 In vitro enzymatic digestion reaction of spCas9 / gRNA (1) Mix the following ingredients in a 200µl PCR tube. Table 7. spCas9 / gRNA in vitro enzyme digestion reaction system .

[0050] 37℃ for 5 minutes, 4℃ forever.

[0051] (2) Mix the following system in a PCR tube. Table 8. CRISPR / Cas9 reaction system .

[0052] (3) After mixing thoroughly, place the test tube into the PCR instrument and run the following program: 37℃, 1hr; 80℃, 5min; 4℃, forever; (4) Add an appropriate volume of DNA lading buffer and run all samples on a 1.5% agarose gel. (5) Analysis results are as follows Figure 1 As shown, the designed sgRNAs all had a cleavage efficiency greater than 50%. Based on the location of their target sites in the genome, gRNA1 (90%), gRNA4 (60%), and gRNA7 (80%) were finally selected to construct gene knockout plasmids.

[0053] Example 2. CRISPR-Cas9-sgRNA Design and Vector Construction 2.1 Design of sgRNA To clone the target sequence into LentiGRISPR-v2 (Addgen, Plasmid #52961, see Sanjana et al Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038 / nmeth.3047.), the following two forms of oligonucleotide chains were synthesized (the prefix CACCG and the suffix CAAA are sticky ends generated after cleavage by the BsmbI restriction endonuclease, and the tandem N represents the sgRNA sequence).

[0054] Oligo 1: 5'-CACCGNNNNNNNNNNNNNNNNN -3' Oligo2: 3'-CNNNNNNNNNNNNNNNNNNNCAAA-5' Therefore, the sgRNA Oligo design is as follows: Table 9. sgRNA Oligo Sequence Design .

[0055] 2.2 Construction of LentiGRISPR-v2 vector 2.2.1 The LentiCRISPR vector was digested with BsmBI at 37°C for 30 min. The following mixture was then placed in a PCR tube, briefly vortexed, and centrifuged.

[0056] Table 10. Enzyme digestion of LentiCRISPR vector .

[0057] The LentiCRISPR-v2 vector is 14,873 bp in size. After enzyme digestion, it produces two fragments: the larger fragment is the target fragment, and the smaller fragment is 1885 bp in size. The target fragment is recovered and then gel recovered.

[0058] 2.2.2 Oligo annealing to double-stranded DNA (1) Mix the following system in a PCR tube, briefly vortex and centrifuge.

[0059] Table 11. Oligo Annealing Reaction System .

[0060] Annealing: Pour boiling water into an open insulated box, place the PCR tubes in the float, and slowly cool to room temperature for more than 3 hours.

[0061] (2) Dilute the annealed product at a ratio of 1:200 in sterile water or EB.

[0062] 2.2.3 Ligation reaction of target sequence and enzyme digestion vector: Mix the following system in a PCR tube and incubate at room temperature for 10 min.

[0063] Table 12. Ligation reaction system of target sequence and enzyme-digested vector .

[0064] 2.2.4 Transformation and Sequencing (1) The bacteria were transformed using fast-transformation competent cells, and the bacterial culture was sent for sequencing. The sgRNA sequences of the three genes are shown in SEQ ID NO.1, 4, and 7. (2) Plasmid sequencing results are as follows Figure 2-4 As shown, the EcoRI single enzyme digestion verification results are as follows: Figure 5 As shown.

[0065] Example 3. Construction of stable transfected cell lines with multiple gene knockouts 3.1 Cell electroporation of plasmids 3.1.1 Plasmid transfer via electroporation (1) Cell seeding and culture: Prepare cells with a density of 4×10⁶ cells / year using complete culture medium (DMEM + 20% FBS). 5 A suspension of porcine ear fibroblasts at a concentration of / mL was seeded into four T25 culture flasks. The flasks were cultured at 37℃ for 16-24 hours until the cell confluence reached 60-70%. (2) Mix the kit combination. The total amount of plasmid added to each system is no more than 1µg. The mixed plasmids are divided into RAG2 plasmid 0.4µg + RAG1 plasmid 0.6µg and RAG2 plasmid 0.4µg + IL2RG plasmid 0.6µg. (3) Digest the cells, resuspend the cells in 1 mL of culture medium for every two bottles, centrifuge at 2500 rpm for 3 min, wash once with PBS, remove the supernatant, and resuspend the cells in the above two plasmid transfection reagent mixture. (4) Transfer to the matching electrode cup; (5) Select the program, choose P3 as the cell type, and manually enter the EN150 program; (6) Insert the electroshock cup and press the Start button; (7) Resuspend the culture medium in the electroporation cup and transfer it entirely to a petri dish.

[0066] 3.2 Screening of immunodeficient cells 3.2.1 After 72 hours of infection, puromycin was added for pressure screening. The optimal concentration of puromycin for screening was 2 μg / mL. 3.2.2 After two weeks of pressurization, switch to normal culture medium; 3.2.3 Two weeks later, the clones were transferred from the petri dish to a 96-well plate using a cloning loop; after confluence, they were transferred to a 48-well plate, then to a 24-well plate, and after confluence, they were digested and resuspended in 1 mL, and 50 µL of the resuspended clones were used for DNA identification.

[0067] 3.3 Identification of Immunodeficient Positive Clones 3.3.1. Trace Genome Extraction (1) Preparation of vector RNA: Add 310µL buffer AE to a test tube containing 310µg of lyophilized vector RNA to obtain a 1μg / ul solution, divide into equal portions, and store at -20℃ (up to 3 freeze-thaw cycles). (2) Transfer 1-100µL of cells into a 1.5ml microcentrifuge tube. Add buffer ATL to a final volume of 100µL; (3) Add 10µL proteinase K, add 100µL buffer AL (add 1µL vector RNA per 100µL), cover and vortex for 15s; (4) Incubating at 56℃ for 10 minutes and shaking can increase DNA yield; (5) Briefly centrifuge the 1.5 ml centrifuge tube to remove any droplets from the cap; (6) Add 50µL of ethanol (96%-100%), close the lid, vortex for 15s, and leave at room temperature for 3min (if the temperature exceeds 25°C, cool on ice before adding the ethanol); (7) Briefly centrifuge the 1.5ml centrifuge tube to remove any droplets from the cap; (8) Carefully transfer the lysis buffer to the QM column (in a 2 mL collection tube, being careful not to wet the edges). Cap the column and centrifuge at 8000 rpm for 1 min. Place the QM column into a clean 2 mL collection tube and discard the collection tube containing liquid. If the lysis buffer does not completely pass through the membrane after centrifugation, centrifuge again at a higher speed until the QM column is empty. (9) Open the QM column and add 500µL of buffer AW1 without wetting the edges. Centrifuge at 8000rpm for 1min, then transfer the QM column to a clean collection tube and discard the collection tube containing liquid. (10) Carefully open the QM column, add 500µL of buffer AW2 without wetting the edges, centrifuge at 8000rpm for 1min, and place the QM column into a clean collection tube. Remove the QM column and collection tube from the centrifuge carefully to avoid contact between the flowing liquid and the QM column; (11) Centrifuge at 14,000 rpm for 3 min to completely dry the membrane. This step is essential, as ethanol carried into the eluent may interfere with some downstream applications. (12) Place the QM column into a clean 1.5ml centrifuge tube. Carefully open the QM column cap. Add 20-100µL of buffer AE or distilled water to the column membrane. If high pH or EDTA affects downstream applications, elute with water. (13) Cover the container and let it stand at room temperature (15-25℃) for 1 min. Then centrifuge at full speed of 14000 pm for 1 min (incubating the QM column containing AE or water at room temperature for 5 min before centrifugation can increase DNA yield).

[0068] 3.3.2 PCR sequencing for screening immune knockout cells (1) The target fragment was amplified using the above monoclonal cell genome as a template. The primer sequences for the target fragment are as follows: The sequences of RAG1-F1 and RAG1-R1 are shown in SEQ ID NO.15 and 16, the sequences of RAG2-F1 and RAG2-R1 are shown in SEQ ID NO.21 and 22, and the sequences of IL2RG-F1 and IL2RG-R1 are shown in SEQ ID NO.27 and 28.

[0069] (2) Mix the following system in a PCR tube, briefly vortex and centrifuge.

[0070] Table 13. PCR amplification system .

[0071] (3) Place the test tube into the PCR instrument and run the following program: 95℃ for 3 min, (95℃ for 20 sec, 58℃ for 30 sec, 72℃ for 3 min) × 29 cycles, 72℃ for 5 min, 4℃ for ∞.

[0072] (4) Perform TA cloning and sequencing on the PCR products. The sequencing results are as follows: Figure 6-7 As shown, RAG1 and RAG2 double gene knockout clones and RAG2 and IL2RG double gene knockout clones were obtained.

Claims

1. An sgRNA, characterized in that, The sgRNA targets the 5222-5241 base range of the RAG-1 gene in porcine fibroblasts, or The sgRNA targets the 4855-4874 base range of the RAG-2 gene in porcine fibroblasts, or The sgRNA targets the IL2RG gene of porcine fibroblasts from base 4489 to base 5224.

2. The sgRNA according to claim 1, characterized in that, The sequence of the sgRNA is shown in SEQ ID NO.1, or in SEQ ID NO.4, or in SEQ ID NO.

7.

3. A vector containing the sgRNA of claim 1 or 2.

4. The carrier according to claim 3, characterized in that, The vector is a CRISPR / Cas9 vector.

5. The carrier according to claim 4, characterized in that, The vector is LentiGRISPR-v2.

6. A method for knocking out the RAG-1 gene, RAG-2 gene, and / or IL2RG-2 gene in porcine fibroblasts using the vector described in claim 4, characterized in that, The method includes the following steps: (1) Transfecting the vector of claim 4 into porcine fibroblasts; (2) Pig fibroblasts with gene knockout obtained from the vector through resistance screening of the pig fibroblasts.

7. The method according to claim 6, characterized in that, The vector in step (1) includes a vector containing sgRNA with the sequence shown in SEQ ID NO.1 and a vector containing sgRNA with the sequence shown in SEQ ID NO.4, or the vector includes a vector containing sgRNA with the sequence shown in SEQ ID NO.4 and a vector containing sgRNA with the sequence shown in SEQ ID NO.

7.

8. A porcine fibroblast that has undergone double gene knockout, characterized in that, The double gene knockout refers to the double gene knockout of RAG-1 and RAG-2 genes, or the double gene knockout of RAG-2 and IL2RG genes.

9. The porcine fibroblasts that have undergone double gene knockout according to claim 8, characterized in that, The porcine fibroblasts that underwent double gene knockout were obtained using the method described in claim 6.

10. The use of the porcine fibroblasts according to claim 8 or 9 in the preparation of immunodeficient pigs, characterized in that, The application involves transplanting the porcine fibroblasts into the mother pig via somatic cell nuclear transfer.

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