Technical method for preparing CD163 gene editing pig by using CRISPR / Gs12-7MAX system

Abstract

The invention discloses a method for preparing a CD163 gene editing pig based on a CRISPR (clustered regularly interspaced short palindromic repeats) / Gs12-7MAX system. The method specifically comprises two editing strategies: one is to edit a sixth intron and a seventh intron of a CD163 gene to realize accurate deletion of a seventh exon so as to obtain a porcine fibroblast monoclonal cell with homozygous deletion of the seventh exon; and 2, editing aiming at a seventh exon of the CD163 gene, and inducing the exon to generate frame-shift mutation to obtain a gene editing pig. The technical system established by the invention has the advantages of high editing efficiency, strong accuracy and simplified operation process, provides a new effective strategy for prevention and control of PRRSV (porcine reproductive and respiratory syndrome), and has important popularization and application values in creation of new materials for pig disease-resistant breeding.

Description

Technical Field

[0001] This invention belongs to the field of animal gene editing and disease-resistant breeding technology, specifically involving a method for efficient and precise editing of the pig CD163 gene based on the CRISPR / Gs12-7 MAX system, as well as the technical system and application of using this method to prepare CD163 gene-edited pigs. Background Technology

[0002] CRISPR / Cas gene editing systems, due to their simplicity and high targeting capabilities, have been widely applied in functional genomics research, animal and plant trait improvement, and agricultural breeding. In pig disease resistance breeding, the CD163 gene, as a key cellular receptor for porcine reproductive and respiratory syndrome virus (PRRSV, commonly known as blue ear disease virus), has become an important target for disease resistance breeding. Editing this gene using the CRISPR / Cas system can block viral invasion, thereby cultivating pig herds resistant to PRRSV, which is one of the core directions in current pig disease resistance breeding.

[0003] Existing technologies demonstrate that editing the porcine CD163 gene using the CRISPR / Cas9 system (e.g., deleting its SRCR5 domain) can produce gene-edited pigs with growth performance and physiological indicators indistinguishable from ordinary pigs, and complete resistance to PRRSV. This provides a feasible technical solution for the control of porcine reproductive and respiratory syndrome (PRRS). However, the traditional CRISPR / Cas9 system has the following inherent limitations: First, its activity is heavily dependent on specific prototype spacer adjacent motifs (PAMs, usually NGGs), resulting in limited editable sites in the genome and difficulty in covering all ideal target regions; second, the system's efficiency in complex editing scenarios such as simultaneous multi-site cutting remains insufficient, limiting its potential in breeding applications such as simultaneous multi-gene editing or functional domain deletion.

[0004] To overcome the aforementioned technological bottlenecks, next-generation CRISPR systems are constantly being developed. Among these, through the mining of metagenomic resources and rational protein design, various Cas protein variants with improved properties have been obtained. The Gs12-7MAX (Chinese Patent No.: ZL202411320266.4) involved in this invention is an engineered, high-fidelity PcuCas12a mutant. Compared with known Cas12a proteins, this variant possesses two significant advantages: higher genome editing activity and lower off-target effects, demonstrating outstanding potential in precise genome editing of plants and animals.

[0005] However, although CRISPR / Cas9 has been applied in pig CD163 editing, a technical solution for efficient editing of the pig CD163 gene and preparation of gene-edited pigs based on the CRISPR / Gs12-7MAX system is still lacking. Existing technologies have not yet disclosed: (1) the editing efficiency and large-fragment editing capability of the Gs12-7MAX system for the pig CD163 gene; and (2) a stable and reproducible process for efficiently preparing CD163 gene-edited pigs using this system. Therefore, developing a method for pig CD163 gene editing and disease-resistant pig breeding based on the CRISPR / Gs12-7MAX system can not only overcome the target limitations and efficiency bottlenecks of traditional systems, providing a better solution for the prevention and control of porcine reproductive and respiratory syndrome (PRRS), but also help improve the agricultural animal gene editing breeding technology system, possessing significant industrial application value. Summary of the Invention

[0006] To overcome the technical bottlenecks of existing CRISPR gene editing tools in pig breeding applications, such as limited editing efficiency and high off-target risk, this invention provides a complete technical solution for efficient and precise editing of the pig CD163 gene based on the CRISPR / Gs12-7MAX system, and for large-scale production of gene-edited pigs.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] Option 1: A method for preparing edited cells with deletion of the seventh exon (Exon 7) of the porcine CD163 gene based on the CRISPR / Gs12-7MAX system, comprising the following steps:

[0009] 1. crRNA design and screening: Highly active crRNAs were designed and screened for the sixth and seventh introns of the porcine CD163 gene, and their sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0010] 2. Vector construction: Construct a co-expression vector containing the Gs12-7MAX coding sequence and the crRNA expression cassette.

[0011] 3. Cell transfection and screening: The co-expression vector was introduced into target cells, and edited cells with homozygous deletion of CD163 gene Exon 7 were obtained through screening.

[0012] Option 2: A method for preparing gene-edited pigs with a frameshift mutation in the seventh exon of the CD163 gene based on the CRISPR / Gs12-7MAX system, comprising the following steps:

[0013] 1. crRNA design and screening: Highly active crRNAs were designed and screened for the seventh exon 7 of the porcine CD163 gene, and their sequences are shown in SEQ ID NO.3.

[0014] 2. Vector construction: Construct a co-expression vector containing the Gs12-7MAX coding sequence and the crRNA expression cassette.

[0015] 3. Donor cell preparation and cloning: The co-expression vector was introduced into porcine somatic cells, and the edited mixed cell population was collected and directly used as donor cells for somatic cell nuclear transfer (SCNT). After embryo transfer, gene-edited pigs with exon 7 frameshift mutation of CD163 gene were obtained.

[0016] Preferably, the co-expression vector is constructed by homologous recombination of a vector backbone, a U6-crRNA scaffold fragment, and a CMV-Gs12-7MAX fragment, and a selected crRNA is inserted therein. The sequence of the U6-crRNA scaffold fragment is shown in SEQ ID NO.4.

[0017] Based on the above method, the present invention also provides:

[0018] 1. Porcine fibroblast monoclonal cells with CD163 gene exon 7 deletion obtained by Scheme 1.

[0019] 2. Gene-edited pigs with a frameshift mutation in the seventh exon of the CD163 gene obtained by scheme 2.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. Editing efficiency and accuracy have been significantly improved.

[0022] The CRISPR / Gs12-7MAX system provided by this invention, through the synergistic effect of the Gs12-7MAX protein and screened highly active crRNA, can precisely target the sixth, seventh, and seventh exon regions of the porcine CD163 gene, creating large DNA double-strand breaks at the target sites, thereby achieving efficient and precise editing of this gene fragment. Compared with existing tools, this system significantly improves the success rate and accuracy of gene editing.

[0023] 2. The process flow is simplified and efficiency is improved.

[0024] This invention establishes a standardized procedure for CRISPR / Gs12-7MAX-based pig CD163 gene editing and the preparation of gene-edited pigs. By directly using electrotransfected pig fibroblasts as somatic cell nuclear transfer donors, the cumbersome monoclonal screening step is eliminated, significantly shortening the experimental cycle and simplifying the operation process, while improving gene editing and cloning efficiency. This method provides an efficient and reproducible technical solution for rapidly constructing gene-edited pigs with specific disease resistance traits, and also provides an important tool for related gene function research and disease model construction. Attached Figure Description

[0025] Figure 1: Schematic diagram of the construction of the Gs12-7MAX protein co-expression vector (PX330-U6-crRNA-Gs12-7MAX-puro), showing the assembly relationship of the vector components, including the PX330 vector backbone, U6-crRNA scaffold and CMV-Gs12-7MAX gene coding region sequence.

[0026] Figure 2: Detection of the editing efficiency of crRNA targeting the sixth and seventh introns of the porcine CD163 gene in porcine cells, and evaluation of the cleavage activity of crRNA at the target site.

[0027] Figure 3: The editing activity of crRNA targeting the sixth intron (crIN6-4) and the seventh intron (crIN7-4) of the porcine CD163 gene was detected by T7 endonuclease I (T7ENI) digestion and agarose gel electrophoresis, demonstrating its ability to perform site-directed mutations at the target site.

[0028] Figure 4: Agarose gel electrophoresis image of CD163 gene amplification products of porcine fibroblast monoclonal cells edited by the CRISPR / Gs12-7MAX system, used to screen positive monoclonal cells with CD163 gene exon 7 deletion.

[0029] Figure 5: Genotypic structure of two porcine fibroblast monoclonal cells with homozygous deletion of the seventh exon of the CD163 gene. Two deletion types are shown: ① deletion of bases from position 12531 (sixth intron) to position 6 (eighth exon); ② deletion of bases from position 12530 (sixth intron) to position 3 (eighth exon).

[0030] Figure 6: Results of the editing efficiency of crRNA targeting the seventh exon of the porcine CD163 gene in porcine cells, assessing its activity in inducing frameshift mutations in this exon.

[0031] Figure 7: The left side shows the genotype analysis of the CD163 gene exon 7 frameshift mutation gene-edited pig, including the sequencing peak diagram and the structural diagram of the deletion of bases 139-199; the right side shows the physical appearance of the gene-edited pig, which together confirm the successful preparation of the gene-edited pig and the realization of the target gene mutation type. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Experimental methods not specified with specific conditions in the embodiments are generally carried out according to conventional experimental methods in the field of molecular biology, or according to the conditions recommended by the manufacturer. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0033] Key material descriptions:

[0034] 1. Lenti-U6-crRNA-puro-zsgreen: A crRNA expression vector constructed in our laboratory. PCR amplification was performed using the direct repeat (DR) sequence and homologous arms at both ends of the CRISPR / Cas12a system as templates. The resulting vector was cloned via homologous recombination into the pKLV2-U6gRNA5(BbsI)-PGKpuro2AZsG-W vector (Addgene #67975), which had been double-digested with BbsI and BamHI, for constitutive expression of crRNA.

[0035] 2. Lenti-CMV-Gs12-7MAX-puro: A Gs12-7MAX protein expression vector constructed in our laboratory. The LentiV_Cas9_puro vector (Addgene #108100) was double-digested with AgeI and BamHI to remove Cas9. The Gs12-7MAX coding sequence was then inserted downstream of the CMV promoter via homologous recombination for stable expression of the Gs12-7MAX protein in mammalian cells.

[0036] 3. PK15-Gs12-7MAX stable cell line: The PK15 cell line stably expressing Gs12-7MAX protein was constructed and preserved in our laboratory for related functional verification experiments.

[0037] 4. Lentiviral packaging plasmids: pMD2.G (Addgene #12259), encoding the VSV-G envelope protein; pSPAX2 (Addgene #12260), encoding the HIV-1 gag / pol protein. The two are used together to produce third-generation lentiviral particles.

[0038] Example 1: Design and construction of a eukaryotic expression vector tandemly containing Gs12-7MAX and crRNA

[0039] To construct a eukaryotic co-expression vector (named pX330-U6-crRNA-Gs12-7MAX-puro) capable of simultaneously expressing Gs12-7MAX protein and a specific crRNA, this embodiment proceeded according to the following steps:

[0040] 1. Template and primers

[0041] Using a chemically synthesized oligonucleotide chain U6-crRNA scaffold (sequence shown in SEQ ID NO.4, synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd.) as a template, the U6-crRNA scaffold expression unit was amplified. Using the Lenti-CMV-Gs12-7MAX-puro vector stored in our laboratory as a template, the CMV-Gs12-7MAX expression cassette was amplified.

[0042] 2. PCR amplification and fragment recovery

[0043] Using the primers listed in Table 1, PCR amplification was performed on the templates described above. After separation of the amplification products by agarose gel electrophoresis, the target bands were excised and recovered to obtain high-purity U6-crRNA scaffold fragments and CMV-Gs12-7MAX fragments (see Table 1). Figure 1 ).

[0044] Table 1 Primer sequence information used in this embodiment

[0045]

[0046] 3. Carrier assembly

[0047] First, the vector pX330-U6-Chimeric was simultaneously mediated using the restriction endonucleases AflIII and EcoRI. _ BB-CBh-hSpCas9 (PX330, Addgene #42230) was double-digested, and the product was purified and recovered. Subsequently, the double-digested PX330 vector DNA and the PCR-recovered product were placed at 50°C for ligation using homologous recombinase. Specific digestion and recombination reaction conditions are detailed in Tables 2 and 3.

[0048] Table 2. Vector double enzyme digestion reaction system

[0049]

[0050] Table 3 Homologous recombination linkage reaction system

[0051]

[0052] Next, 10 μL of the recombinant product was added to 30 μL of DH-5α competent cells and incubated on ice for 30 min. Then, the cells were heat-shocked in a 42℃ water bath for 45 s, followed immediately by an ice bath for 5 min. Antibiotic-free LB medium was added to bring the volume to 1 mL, and the cells were incubated at 37℃ with shaking for 1.5 h. The cells were centrifuged at 3000 r / min for 1 min, and a portion of the supernatant was discarded, retaining approximately 150-200 μL of bacterial culture. This bacterial culture was evenly spread on LB agar plates containing ampicillin and incubated upside down at 37℃ for 12-16 h. After incubation, single colonies were picked for sequencing verification. After confirming the sequence was correct, positive clones were selected for further expansion culture, and plasmids were extracted using an endotoxin removal kit. Finally, the Gs12-7MAX co-expression vector with crRNA was successfully constructed and named PX330-U6-crRNA-Gs12-7MAX-puro.

[0053] Example 2: Construction of a monoclonal strain of porcine fetal fibroblasts with homozygous knockout of the seventh exon of the CD163 gene using the Gs12-7MAX system

[0054] In this embodiment, to screen for highly active crRNAs, four specific crRNAs were first designed using CRISPR-offinder software (http: / / www.biootools.com) targeting introns six and seven of the porcine CD163 gene (Ensembl: ENSSCG00000033146). Subsequently, specific primers were designed on the flanking sequences of each crRNA using Primer Premier 5.0 software for subsequent crRNA activity amplification and detection. These primers were synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd. (specific sequences are shown in Table 4).

[0055] 5 μL each of the synthesized crRNA primers F and R (both 10 μmol / L) were mixed and annealed in a PCR instrument using the following program: 95℃ for 10 min; 65℃ for 60 min. The annealed product was ligated into the Lenti-U6-crRNA-puro-zsgreen vector, which had been linearized by BbsI (NEB, Beijing). After transformation, plating, and overnight incubation, single colonies were picked the following day for sequencing verification. After confirming the sequence was correct, positive clones were selected for further amplification culture, and plasmids were extracted using an endotoxin removal kit. The eight plasmids obtained were named as follows: Lenti-U6-crIN6-1-puro-zsgreen, Lenti-U6-crIN6-2-puro-zsgreen, Lenti-U6-crIN6-3-puro-zsgreen, Lenti-U6-crIN6-4-puro-zsgreen, Lenti-U6-crIN7-1-puro-zsgreen, Lenti-U6-crIN7-2-puro-zsgreen, Lenti-U6-crIN7-3-puro-zsgreen, and Lenti-U6-crIN7-4-puro-zsgreen.

[0056] Table 4. Information on crRNA and primer sequences targeting the sixth and seventh introns of the CD163 gene.

[0057]

[0058] Subsequently, the editing activity of crRNA targeting introns six and seven of the porcine CD163 gene was evaluated. HEK293T cells were co-transfected with pMD2.G:psPAX2:Lenti-U6-INcrRNA-puro-zsgreen at a mass ratio of 1:2:3, packaged with lentivirus, and the resulting virus was used to infect the PK15 cell line (PK15-Gs12-7MAX) that stably expresses Gs12-7MAX. Genomic DNA was then extracted from the cells according to the instructions of the Tiangen DNA Extraction Kit (KG203), and PCR amplification was performed targeting the region. The amplified product was purified using a DNA purification kit (Novizan, Nanjing) and divided into two parts: one part was sent to Beijing Aoke Dingsheng Biotechnology Co., Ltd. for sequencing analysis, and the other part was used for T7ENI (NEB, Beijing) enzyme digestion (enzyme digestion conditions are detailed in Tables 5-6).

[0059] By comparing sequencing results and detecting the gel electrophoresis bands of enzyme digestion products, it was found that crIN6-4 and crIN7-4 can effectively cleave target regions in the genome. Figure 2 , Figure 3 Therefore, crIN6-4 and crIN7-4 were subsequently selected to prepare positive clones of fibroblasts with deletion of the seventh exon of the CD163 gene.

[0060] Table 5 T7ENI enzyme digestion system

[0061]

[0062] Table 6 T7ENI Annealing Procedure

[0063]

[0064] Note: Add 1 μL of T7ENI to the annealed product and incubate at 37°C for 15 min.

[0065] To construct a knockout vector targeting introns six and seven of the porcine CD163 gene, crIN6-4 and crIN7-4 were tandemly synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd., and named crIN6-7 (sequence shown in SEQ ID NO.5). Subsequently, crIN6-7 was ligated with the linearized vector PX330-U6-crRNA-Gs12-7MAX-puro, which had been digested with SapI (NEB, Beijing), to construct an expression vector capable of simultaneously expressing two crRNAs, named PX330-U6-crIN6-7-Gs12-7MAX-puro.

[0066] Resuscitated porcine fetal fibroblasts were seeded into 10 cm culture dishes. When the cell confluence reached approximately 90%, electroporation was performed using an electroporator (Celetrix, USA). The electroporation system was as follows: porcine fetal fibroblasts were resuspended in 100 μL of electroporation buffer (Celetrix, USA), and the cell density was adjusted to approximately 5 × 10⁻⁶ cells / mL. 7 Add 10 μg of PX330-U6-crIN6-7-Gs12-7MAX-puro plasmid to cells / mL and electrolyze once at 520 V.

[0067] After electroporation, cells were transferred to 6-well plates for further culture. After 72 h of electroporation, puromycin was added to the culture medium to a final concentration of 2 μg / mL. The medium was replaced with fresh puromycin every 48 h, and this process was repeated for 3-4 days until all cells in the control group (without plasmids) died. Surviving cells were collected; a portion was used for genomic DNA extraction and genotyping, while the remaining cells were plated at low density in 10 cm culture dishes.

[0068] After cell clones appeared in the culture dish, 132 single-clone cells were picked using a cloning loop (Corning, USA) and transferred to 24-well plates for further culture. PCR amplification was performed on the 72 surviving cell lines. The results are as follows: Figure 4 As shown: Wild-type cells amplified genomic DNA yielded a single band (1328 bp); gene-edited heterozygous cells amplified two bands (1328 bp and 590-600 bp); gene knockout homozygous cells amplified a single band of varying sizes, ranging from 590 to 600 bp. A total of 20 heterozygous cell lines and 2 homozygous clones (Clone 2 and 12) were identified, with a positive rate of 16.6%. Figure 4 Further sequencing of the amplified bands in monoclonal cells 2 and 12 revealed that, compared to the wild type, the two cell lines were missing 733 bp and 731 bp, respectively. Figure 5 The cells were identified as porcine fetal fibroblast monoclonal cells with homozygous deletion of the seventh exon of the CD163 gene.

[0069] In summary, the Gs12-7MAX / crRNA co-expression plasmid system can be used to successfully achieve precise deletion of the seventh exon of CD163 and obtain the corresponding monoclonal fibroblasts.

[0070] Example 3: Constructing a CD163 gene exon 7 frameshift mutant gene-edited pig using the Gs12-7MAX system

[0071] In this embodiment, a highly active crRNA targeting exon 7 of the porcine CD163 gene was first screened and constructed. Four specific crRNAs targeting this exon were designed using CRISPR-offinder software (crRNA sequences are shown in Table 7). Each crRNA was ligated into the Lenti-U6-crRNA-puro-zsgreen vector, which was linearized by BbsI (NEB, Beijing), and transformed into DH-5α competent cells. Single colonies were picked and identified after 12-16 h. Colonies with correct sequencing were selected for amplification culture, and plasmids were extracted using an endotoxin-free kit. The four obtained plasmids were named Lenti-U6-crEN7-1-puro-zsgreen, Lenti-U6-crEN7-2-puro-zsgreen, Lenti-U6-crEN7-3-puro-zsgreen, and Lenti-U6-crEN7-4-puro-zsgreen, respectively.

[0072] HEK293T cells were seeded in 10 cm culture dishes and incubated overnight at 37°C. When the cell density reached approximately 80%, HEK293T cells were co-transfected with pMD2.G: psPAX2: Lenti-U6-EncrRNA-puro-zsgreen at a mass ratio of 1:2:3, followed by lentiviral packaging. The resulting virus was then used to infect PK15-Gs12-7MAX monoclonal cells. Genomic DNA was extracted from the cells 3 days after infection, and the target region was amplified by PCR and sequenced. The results are as follows: Figure 6 As shown, the editing efficiency of crEN7-2 reached 82%, therefore crEN7-2 was selected for the subsequent preparation of CD163 gene-edited pigs.

[0073] Table 7. crRNA sequence information targeting the seventh exon of porcine CD163

[0074]

[0075] Next, crEN7-2 was ligated into the linearized vector PX330-U6-crRNA-Gs12-7MAX-puro, which had been digested with SapI (NEB, Beijing), to obtain the expression vector PX330-U6-crEN7-2-Gs12-7MAX-puro. This plasmid was then electrotransfected into porcine fetal fibroblasts. Three days after transfection, the co-edited cells were directly collected as donor cells for subsequent somatic cell cloning and embryo transfer.

[0076] A total of 250 embryos were transferred to each of three surrogate sows. Ultrasound examination confirmed successful pregnancy in all three sows, and cloned piglets were delivered naturally on day 118. Ear tissue samples were collected from the piglets, genomic DNA was extracted, and target regions were amplified and genotyped. Sequencing results showed a 58 bp deletion in the CD163 gene in one of the piglets. Figure 7 This deletion can lead to frameshift mutations in genes.

[0077] The above results demonstrate that CRISPR / Gs12-7MAX technology can be used to successfully prepare gene-edited pigs resistant to porcine reproductive and respiratory syndrome (PRRS) with a frameshift mutation in the seventh exon of the CD163 gene, providing a new technical pathway for the preparation of gene-edited pigs.

[0078] In summary, this invention, based on the CRISPR / Gs12-7MAX gene editing system, achieved the editing of the seventh exon of the porcine CD163 gene through two different strategies: first, obtaining porcine fetal fibroblast monoclonal cells with homozygous deletion of this exon fragment; and second, successfully preparing gene-edited pigs with frameshift mutations in the seventh exon. This research lays the technical foundation for the application of the CRISPR / Gs12-7MAX system in porcine disease-resistant breeding.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing edited cells with deletion of the seventh exon of the porcine CD163 gene based on the CRISPR / Gs12-7MAX system, characterized in that: The method includes screening for highly active crRNAs targeting introns six and seven of the CD163 gene, with crRNA sequences shown in SEQ ID NO.1 and SEQ ID NO.

2. A co-expression vector of Gs12-7MAX and crRNA is constructed, and the co-expression vector is transformed into cells to screen for edited cells with homozygous deletion of the seventh exon of the CD163 gene.

2. A method for preparing edited cells with a frameshift mutation in the seventh exon of the CD163 gene based on the CRISPR / Gs12-7MAX system, characterized in that: The process includes screening for highly active crRNA targeting exon 7 of CD163, with the crRNA sequence shown in SEQ ID NO.3; constructing a co-expression vector of Gs12-7MAX and crRNA; and transforming the co-expression vector into cells to screen for edited cells with a mutation in the seventh outer frameshift of the CD163 gene.

3. A method for preparing edited pigs with a frameshift mutation in the seventh exon of the CD163 gene based on the CRISPR / Gs12-7MAX system, characterized in that: The process includes screening for highly active crRNA targeting exon 7 of the CD163 gene, with the crRNA sequence shown in SEQ ID NO.3; constructing a co-expression vector of Gs12-7MAX and crRNA; transducing the co-expression vector into cells; collecting the mixed-edited cells and using them directly as donor cells for somatic cell cloning and embryo transfer to obtain edited pigs with frameshift mutations in exon 7 of the CD163 gene.

4. The method according to any one of claims 1 to 3, characterized in that, The cells in question are porcine fetal fibroblasts.

5. The method according to any one of claims 1 to 3, characterized in that, The co-expression vector is constructed by homologous recombination of a vector backbone, a U6-crRNA scaffold fragment, and a CMV-Gs12-7MAX fragment, and a selected crRNA is inserted. The sequence of the U6-crRNA scaffold fragment is shown in SEQ ID NO.

4.

6. Edited cells with deletion of the seventh exon of the porcine CD163 gene prepared by the method of claim 1.

7. Edited cells with a frameshift mutation in the seventh exon of the CD163 gene, prepared by the method of claim 2.

8. The application of the method described in claim 3 in the breeding of gene-edited pigs resistant to porcine reproductive and respiratory syndrome (PRRS).

9. The application of the porcine CD163 gene-edited cells as described in claim 6 or 7 in the breeding of gene-edited pigs resistant to porcine reproductive and respiratory syndrome (PRRS).

Images