CRISPR / Cas13d system for inhibiting foot and mouth disease virus and application

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a CRISPR / Cas13d system for inhibiting foot-and-mouth disease virus and application of the CRISPR / Cas13d system. According to the invention, 44 crRNAs sequences and one control sequence are designed in total in a targeted FMDV whole genome, crRNAs for inhibiting the foot and mouth disease virus are obtained through screening, and the crRNAs are constructed into an expression vector of Cas13d protein carrying a nuclear localization signal, so that a CRISPR / Cas13d system for inhibiting the foot and mouth disease virus is obtained through construction; a transgenic cell line obtained after transfecting cells by the CRISPR / Cas13d system can significantly inhibit the replication of FMDV, can be used for the disease-resistant breeding research of foot-and-mouth disease, provides an important theoretical basis for the creation of new livestock varieties with disease-resistant characteristics, and provides a new idea for the research of disease-resistant breeding of livestock.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a CRISPR / Cas13d system for inhibiting foot-and-mouth disease virus and its application. Background Technology

[0002] In recent years, the CRISPR / Cas system has been widely used in gene editing, molecular therapy, and disease-resistant breeding. This system includes various types and has enormous application potential. CRISPR / Cas13, belonging to type VI of the CRISPR / Cas system, can target and cleave single-stranded RNA, thereby interfering with gene expression at the transcriptional level. The discovery of this system has expanded the application of CRISPR systems in RNA virus interference. The Cas13 family consists of a single Cas13 nuclease and a precursor crRNA. The Cas13 nuclease is activated upon recognizing single-stranded RNA, further cleaving the precursor crRNA into mature crRNA. The protospacer flanking sequence (PFS) recognizes target RNA of 24-30 nt in length. The crRNA pairs complementaryly with the target RNA, guiding the Cas13 nuclease to cleave and degrade the target RNA, thereby inhibiting target gene expression at the transcriptional level. The Cas13 family includes Cas13a, Cas13b, Cas13c, Cas13d, etc. Among them, the average length of Cas13d protein is about 930 amino acids, which is 20% smaller than other Cas13 proteins. It has advantages such as high targeting efficiency, low off-target efficiency and easy delivery by vectors.

[0003] Foot-and-mouth disease (FMD) is an acute, highly contagious, and deadly infectious disease caused by the foot-and-mouth disease virus (FMDV). Susceptible animals include more than 70 species of cloven-hoofed animals, including pigs, cattle, sheep, and African buffalo, causing significant economic losses to my country's livestock industry. During outbreaks, intensively raised cattle and pigs are highly susceptible to infection, especially young animals, with mortality rates reaching 80%-100%. Furthermore, trade in animal products is restricted within affected areas, rendering meat and dairy products unproductive, further exacerbating the losses in the livestock industry. The FMDV viral particle consists of a capsid and genomic RNA. Its genome is a single-stranded positive-sense RNA, approximately 8.4 kb in length, including a 5' untranslated region (5'UTR), an open reading frame (ORF), and a 3' untranslated region (3'UTR). The FMDV encodes four structural proteins (VP1, VP2, VP3, and VP4) and eight non-structural proteins (L...). proFoot-and-mouth disease virus (FMDV) has seven serotypes, including O, A, C, SAT 1, SAT 2, SAT 3, and Asia 1. The lack of effective cross-protection between different serotypes makes the prevention and control of FMDV more difficult.

[0004] The CRISPR / Cas13d system, as an RNA editing tool, has been widely used in viral diagnosis, treatment of RNA knockdown diseases, and treatment of single-base mutation diseases. Several research teams have utilized the CRISPR / Cas13d system for targeted inhibition studies of RNA viruses. For example, in 2020, Bi Dengfeng and his team successfully used the CRISPR / Cas13d system to knock down the SUV39H1 and SUV39H2 genes in porcine fetal fibroblasts, reducing H3K9me3 levels and thereby increasing the developmental rate of porcine nuclear transfer embryos. This established a CRISPR / Cas13d-mediated porcine endogenous gene knockdown platform, laying the foundation for porcine gene function research and providing new insights into improving disease resistance in pigs. In February 2020, Pier Paolo Pandolfi's team proposed using the Cas13d system to combat the COVID-19 pandemic and prevent mutants from spreading the virus. This research reveals the potential applications of the CRISPR / Cas13d system in the field of antiviral research. In March of the same year, Huang Pengyu and Yang Hui's research group successfully silenced the Pcsk 9 gene efficiently in mouse liver using the CasRx system, significantly reducing cholesterol levels in mouse blood. This study confirmed that Cas13d has the activity of targeting and inhibiting RNA in animals, promoting the further development of the CRISPR / Cas13d system. In 2023, Gene W Yeo's team at the University of California made significant progress in a Huntington's disease (HD) mouse model using adeno-associated virus (AAV) to deliver a vector carrying the Cas13d protein and target RNA. This method significantly improved the motor coordination ability of the Huntington's disease mouse model, slowed the degeneration of the striatum in the brain, and reduced the expression of toxic proteins. The results indicate that the CRISPR / Cas13d system shows potential efficacy in treating adult and adolescent HD patients.

[0005] This invention targets foot-and-mouth disease virus, which seriously threatens the livestock industry. It aims to develop a new disease control strategy based on the gene editing technology CRISPR / Ca s13d, providing important support for the genetic eradication of foot-and-mouth disease and offering new ideas for the control of other diseases. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a CRISPR / Cas13d system and its application for inhibiting foot-and-mouth disease virus, providing an important theoretical basis for creating new livestock breeds with disease-resistant characteristics and offering new ideas for livestock disease-resistant breeding. Specifically, it includes the following:

[0007] In a first aspect, the present invention provides crRNAs for targeting the genome of foot-and-mouth disease virus, said crRNAs being selected from at least one of the target sequences shown in SQE ID NO.1-10.

[0008] Preferably, the crRNAs are selected from at least one of the target sequences shown in SQE ID NO.1-4.

[0009] In a second aspect, the present invention provides the use of the crRNAs described in the first aspect in the preparation of drugs or reagents for inhibiting foot-and-mouth disease virus.

[0010] Thirdly, the present invention provides a CRISPR / Cas13d system for inhibiting foot-and-mouth disease virus, wherein the CRISPR / Cas13d system is obtained by linking the crRNAs described in the first aspect above to an expression vector of the Cas13d protein.

[0011] Preferably, the expression vector of the Cas13d protein carries a nuclear localization signal.

[0012] Fourthly, the present invention provides a method for constructing the CRISPR / Cas13d system described in the third aspect above, the method comprising the following steps:

[0013] (1) Add AAAA to the 5' end of the target RNA to obtain a reverse oligonucleotide; at the same time, obtain the corresponding DNA complementary strand according to the target RNA, and add AAAAC to its 5' end to obtain a forward oligonucleotide; synthesize the above forward oligonucleotide and reverse oligonucleotide respectively, and anneal the synthesized forward oligonucleotide and reverse oligonucleotide to form a double strand;

[0014] (2) The double strands obtained in step (1) are ligated into the expression vector of the Cas13d protein to obtain the CRISPR / Cas13d system.

[0015] Preferably, the expression vector of the Cas13d protein is an expression vector of the Cas13d protein carrying a nuclear localization signal.

[0016] Fifthly, the present invention provides a cell line obtained by transfecting a host cell with the gene editing vector described in the fourth aspect above.

[0017] Preferably, the host cell is selected from BHK-21 cells.

[0018] Sixthly, the present invention provides the application of the cell lines described in the fifth aspect above in any of the following aspects:

[0019] (1) Application in screening or preparing drugs that inhibit the proliferation of foot-and-mouth disease virus;

[0020] (2) Application in preparing in vitro screening models for drugs against foot-and-mouth disease virus;

[0021] (3) Application in preparing cell models for studying the replication mechanism of foot-and-mouth disease virus;

[0022] (4) Application in the preparation of cell models for breeding against foot-and-mouth disease virus.

[0023] The beneficial effects of this invention are as follows: First, this invention studies, designs, and screens crRNAs that target and inhibit FMDV; second, this invention constructs the crRNAs into expression vectors carrying nuclear localization signals for the Cas13d protein, thus constructing a CRISPR / Cas13d system for inhibiting foot-and-mouth disease virus; finally, using the CRISPR / Cas13d system to transfect host cells, a CRISPR / Cas13d cell line for inhibiting foot-and-mouth disease virus is obtained. This CRISPR / Cas13d cell line can significantly inhibit FMDV replication and can be used for research on foot-and-mouth disease resistance, providing an important theoretical basis for creating new livestock breeds with disease-resistant characteristics and offering new ideas for livestock disease-resistant breeding. Attached Figure Description

[0024] Figure 1 Schematic diagram of the location of crRNA targeting the FMDV / O / GDBY / 2010 genome sequence;

[0025] Figure 2 A schematic diagram of the pCAG-NLS-Cas13d-HA-NLS-U6crRNA-SV40puro plasmid targeting the FMDV / O / GDBY / 2010 genome sequence;

[0026] Figure 3 Western blot assay was used to detect the expression level of Cas13d protein in the BHK-21 transgenic cell line;

[0027] Figure 4 FMDV infection of BHK-21 transgenic cell line CPE cells for 12 hpi;

[0028] Figure 5 Real-time quantitative PCR was used to detect the viral mRNA level of FMDV-infected BHK-21 transgenic cell line 12 hpi.

[0029] Figure 6Cell viability after Cas13d protein expression. Detailed Implementation

[0030] First, this invention designed 44 crRNA sequences and 1 control sequence targeting the entire FMDV genome, constructed them into an expression vector carrying the Cas13d protein with a nuclear localization signal, and transfected it into BHK-21 cells. After puromycin selection, the positive rate of candidate cells was successfully increased. Second, Western blotting was performed on the aforementioned cells to detect the expression of the Cas13 protein. Subsequently, FMDV was used to infect transgenic positive cells, and the antiviral effect of transgenic positive cells was evaluated by RT-qPCR. The results showed that the expression of the Cas13d protein significantly reduced viral mRNA levels and inhibited FMDV replication, demonstrating that the CRISPR / Cas13d system plays an important role in the anti-FMDV process. Cell viability assays showed that the growth characteristics of transgenic positive cells were not significantly different from wild-type BHK-21 cells, laying the foundation for subsequent antiviral research.

[0031] The FMDV / O / GDBY / 2010 strain described in the following experiments was isolated and preserved by the Foot-and-Mouth Disease and Emerging Disease Epidemiology Team of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; the vector pCX2393 was donated by Researcher Xu Chunlong of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, and we modified it to become pCAG-NLS-Cas13d-HA-NLS-U6-SV40puro; the restriction endonuclease BbsⅠ was purchased from NEB; T4 DNA ligase, Trizol reagent, and 2×ChamQ Universal SYBR qPCR Master Mix were purchased from Novizan Biosciences Co., Ltd.; DH5α competent cells were purchased from Shenzhen Kangti Life Technology Co., Ltd.; and the gel extraction kit and micro DNA extraction kit were purchased from OME. GA (Gibberellins et al.); protein pre-staining markers were purchased from Invitrogen; genomic DNA extraction kits were purchased from Tiangen Biotech Co., Ltd.; DMEM cell culture medium was purchased from Gibco; 0.25% EDTA trypsin was purchased from Gibco; fetal bovine serum was purchased from Ecosai Biotechnology Co., Ltd.; triple antibody and CCK-8 kits were purchased from Beyotime Biotechnology Co., Ltd.; Polyplus jetPRIME transfection reagent was purchased from Polyplus Transfection; mouse anti-β-Actin monoclonal antibody was purchased from Proteintech; mouse anti-HA monoclonal antibody and HRP-labeled goat anti-mouse IgG antibody were purchased from Abmart.

[0032] Example 1: Construction of plasmid pCAG-NLS-Cas13d-HA-NLS-U6crRNA-SV40puro

[0033] By retrieving the FMDV / O / GDBY / 2010 genome sequence from the NCBI database and referring to the information provided on the website designed by Zhang Feng's laboratory (https: / / cas13design.nygenome.org / ), a whole-genome crRNA targeting FMDV / O / GDBY / 2010 (e.g., crRNA targeting the FMDV / O / GDBY / 2010 genome) was designed. Figure 1 (As shown). A total of 44 crRNA sequences and 1 control sequence were designed, and their target sequences are shown in SEQ ID NO.1-45 respectively:

[0034] cr5: CTCCAGGCCCAACAACCACGACAACTGCTG (shown in SEQ ID NO.1);

[0035] cr10: GAGCGTTGGAGTCACTTACGGGTACGCAAC (shown in SEQ ID NO. 2);

[0036] cr14: TCACCAACCTCCTGGACGTGGCTGAGGCTT (shown in SEQ ID NO.3);

[0037] cr31: ACGAGGGACCGGTGAAGAAGCCTGTCGCTT (as shown in SEQ ID NO.4);

[0038] cr19: ACCTTACCTGGGTGCCAAATGGAGCACCTG (as shown in SEQ ID NO.5);

[0039] cr26: TCAAGTACTTCGCCCAGATGGTCTCGACCA (shown in SEQ ID NO. 6);

[0040] cr27: GGACACCCACACCAACCCTGTGGCAATGTT (as shown in SEQ ID NO.7);

[0041] cr29: CACCACAGATGACAAGACTCTTGACGAGGC (as shown in SEQ ID NO. 8);

[0042] cr30: CGCCAGAGACCTCTGAAAGTGAGAGCCAAG (as shown in SEQ ID NO. 9);

[0043] cr32: AGCCCGTTGAGCTCATACTCGACGGGAAGA (shown in SEQ ID NO. 10);

[0044] cr1:TTCACCCACCTGCAGCTGGACTCACGGCAC (shown in SEQ ID NO.11);

[0045] cr2: GGTCGTTAGCGCTGTCCTGGGCACTCCTGC (as shown in SEQ ID NO.12);

[0046] cr3: CCGGCGTTAACGGGATGTAATCACAAGATG (shown in SEQ ID NO. 13);

[0047] cr4: AGCCACGTCTCAGGACCCACCATGTGTGCA (shown in SEQ ID NO. 14);

[0048] cr6: TGGAACATCAAACACTTGCTCCACACCGGG (as shown in SEQ ID NO.15);

[0049] cr7: GGTACGCGATCGACGACGAAGAATTCTACC (as shown in SEQ ID NO.16);

[0050] cr8: CTCCATGGACACCCAACTTGGTGACAATGC (shown in SEQ ID NO. 17);

[0051] cr9: AAGTGGCCAGCTCTGCCTTCAGCGGTCTT (as shown in SEQ ID NO.18);

[0052] cr11: GGAGCTCCCGACTGACCACAAAGGTGTCTA (shown in SEQ ID NO. 19);

[0053] cr12: GACGAACATGACAGCCCACATCAAGGTGCC (shown in SEQ ID NO. 20);

[0054] cr13: AGGCGCTCCGCAGATCAAGGTGTATGCCAA (as shown in SEQ ID NO.21);

[0055] cr15: CCTGCACTTCATGTTCACAGGTCCCACTGA (shown in SEQ ID NO. 22);

[0056] cr16: ACGCAGAGTGGGACACGGGTCTGAACTCAA (as shown in SEQ ID NO.23);

[0057] cr17: GCTGCTGAGACCACAAATGTTCAGGGATGG (shown in SEQ ID NO. 24);

[0058] cr18: CAGAGGCGCCACCACACAGACGTCTCATTC (as shown in SEQ ID NO.25);

[0059] cr20: CGCTGCCTACTTCTTTCAACTACGGTGCCA (as shown in SEQ ID NO.26);

[0060] cr21: GGTTGGTATCCGCGTTTGAGGAATTGGCCA (shown in SEQ ID NO. 27);

[0061] cr22: TCATGCTGGCTGACACCGGTCTTGAGATTC (as shown in SEQ ID NO.28);

[0062] cr23: GCTAGCGGGTTGGTCAAGGTCGCCTCGAG (as shown in SEQ ID NO.29);

[0063] cr24: GCGACTGGATTAAAGCATGGATCGCCTCAG (shown in SEQ ID NO.30);

[0064] cr25: ACGTCCACATTGCCAACCTGTGCAAAGTGG (as shown in SEQ ID NO.31);

[0065] cr28: TCCATGACTCCATCAAGGAAGAGCTCCGAC (shown in SEQ ID NO. 32);

[0066] cr33: TGATCAACAACGCTGACGTCGGGAGACTGA (shown in SEQ ID NO.33);

[0067] cr34: CACATTCATCGTTGGCACTCACTCTGCAGG (as shown in SEQ ID NO.34);

[0068] cr35: GCATCTACGAGGCCATTAAAGGCGTTGACG (shown in SEQ ID NO.35);

[0069] cr36: CCTTCCTGAAGGACGAAATTCGCCCGATGG (as shown in SEQ ID NO.36);

[0070] cr37: TCGTGAACACGGAACACGCCTATGAGAACA (shown in SEQ ID NO. 37);

[0071] cr38: ATCTCCTACGGAGACGACATCGTGGTTGCT (as shown in SEQ ID NO.38);

[0072] cr39: CCTGACTGACTCTTATGCTTACA (as shown in SEQ ID NO.39);

[0073] cr40: ATGGTGCCAGAACTTTGCTCTAT (as shown in SEQ ID NO.40);

[0074] cr41: TGGGTTTGCCTGTTTCAAATTAC (as shown in SEQ ID NO.41);

[0075] cr42: TGTGCTGTACTTTCTCATTGAGA (as shown in SEQ ID NO.42);

[0076] cr43: GTGGTGCGCTCATTGACTTCGAG (as shown in SEQ ID NO.43);

[0077] cr44: GCGGTTGGTTGTAATCCTGATGT (as shown in SEQ ID NO.44);

[0078] crNC: CACACGGTGTGTTCAACCCTGAGTACGGCC (shown in SEQ ID NO. 45).

[0079] AAAA is added to the 5' end of the target RNA to obtain a reverse oligonucleotide; at the same time, the corresponding complementary DNA strand is obtained according to the target RNA, and AAAAC is added to its 5' end to obtain a forward oligonucleotide; the above forward and reverse oligonucleotides are synthesized respectively, and the synthesized forward and reverse oligonucleotides are annealed to form a double-stranded linker.

[0080] The vector pCAG-NLS-Cas13d-HA-NLS-U6-SV40puro was digested with the restriction endonuclease BbsⅠ. The digestion products were separated by 1% agarose gel electrophoresis, and the vector backbone was recovered from the gel. The recovered vector backbone and double-stranded linker were ligated using T4 DNA ligase (e.g., ...). Figure 2 (as shown);

[0081] Finally, the ligation product was transformed with E. coli DH5α competent cells and plated on LB plates containing ampicillin resistance, and incubated overnight at 37°C.

[0082] Single colonies were picked and cultured overnight in a shaker at 37°C in liquid LB medium containing ampicillin resistance. Plasmids were then extracted and named pCAG-NLS-Cas13d-HA-NLS-U6crRNA1-SV40puro to pCAG-NLS-Cas13d-HA-NLS-U6crRNA44-SV40puro and pCAG-NLS-Cas13d-HA-NLS-U6crRNANC-SV40puro, respectively.

[0083] Example 2 Cell Transfection and Drug Screening

[0084] Resuscitate BHK-21 cells and passage them 1-2 times. Once the cells are stable, transfer them to 6-well cell culture plates at a rate of 2 × 10⁶ cells per well. 5 BHK-21 cells were seeded at a density of [number] cells and placed in a cell culture incubator. When the cell density reached 70%-80%, [the cells were] seeded according to the jetP [method / procedure]. The plasmid constructed in Example 1 was transfected using the transfection reagent instructions. Specifically, the pCAG-NLS-Cas13d-HA-NLS-U6crRNA1-SV40puro plasmid was transfected into BHK-21 cells using 1.5 μg of pCAG-NLS-Cas13d-HA-NLS-U6crRNA1-SV40puro and 0.5 μg of PBase. 24 h post-transfection, cells were digested and one-third were seeded into new 6-well plates. Puromycin was added to each well to a final concentration of 4 μg / mL, and the cells were screened in an incubator for 3 days (the screening effect was observed daily during this period) to remove negative cells, thus obtaining transgenic cell lines targeting the entire FMDV genome, hereinafter referred to as cr1-cr44 and crNC.

[0085] Example 3: Western blot assay to detect the expression of Cas13d protein in transgenic cell lines.

[0086] Protein samples were collected from cr1-cr44 and crNC cells for Western blotting experiments to detect the expression of Cas13d protein in each group of cells. Results are as follows: Figure 3 As shown, HA-labeled Cas13d protein was well expressed in cells of all groups, indicating that the cr1-cr44 and crNC transgenic cells were successfully prepared.

[0087] Example 4: FMDV infection of BHK-21 transgenic cell line CPE cells at 12 hpi

[0088] Positive cells transfected with cr1-cr44 and crNC were seeded into new 12-well plates. Once the cell density reached approximately 90%, the culture medium was aspirated, and the cells were washed once with PBS. The cells were then seeded with FMDV / O / GDBY / 2010 (MOI = 0.001). The plates were placed in a cell culture incubator, and the supernatant virus solution was aspirated after 1 hour. The cells were washed twice with PBS, and 1 mL of 2% DMEM medium was added for further culturing.

[0089] FMDV-infected cells showed CPE at 12 hpi, as shown in... Figure 4 As shown, the lesion severity and number of lesion cells in cr5, cr9, cr10, cr14, cr19 and cr26 cells were significantly lower than those in the control group crNC cells, indicating that transgenic positive cells can inhibit FMD V replication.

[0090] Example 5: Real-time quantitative PCR assay to detect FMDV mRNA levels in cells after infection.

[0091] Cells transfected with cr1-cr44 and crNC were seeded into new 12-well plates. Once the cell density reached approximately 90%, the culture medium was aspirated, and the cells were washed once with PBS. The cells were then seeded with FMDV / O / GDBY / 2010 (MOI = 0.001). The plates were placed in a cell culture incubator, and the supernatant virus solution was aspirated after 1 hour. After washing the cells twice with PBS, 1 mL of 2% DMEM medium was added, and the cells were cultured for another 12 hours. Cells were infected with FMDV for 12 hours, and cell and viral RNA samples were collected for relative quantification.

[0092] qPCR primer sequences: BHK-21-GAPDH-qF: tttggccgtatttgacgcct (SEQ ID NO.46); BHK-21-GAPDH-qR: atgggtagagtcatactgga (SEQ ID NO.47); FMDV-3D-qF: CACTGGTCACAGGCTAAGG (SEQ ID NO.48); FMDV-3D-qR: CCCTTCTCAGATTCCGAGT (SEQ ID NO.49).

[0093] The results are as follows Figure 5 As shown, after 12 hpi of FMDV infection, the mRNA levels of FMDV in cr5, cr10, cr14, cr19, cr26, cr27, cr29, cr30, cr31, and cr32 cells were significantly lower than those in the control crNC cells. This indicates that the crRNA designed in this application, represented by SEQ ID NO. 1-10, has a significant effect on reducing viral mRNA levels and inhibiting FMDV replication, demonstrating that the CRISPR / Cas13 system plays an important role in the anti-FMDV process.

[0094] Example 5 Cell viability detection

[0095] Cr5, Cr10, Cr14, Cr31, CrNC, and BHK-21 cells were digested and cultured in 96-well cell culture plates at a density of 1×10⁻⁶ cells / well. 5 -2×10 5 Inoculate with 100 μL of 100 cells / mL into each well. Place the 96-well plate in a cell culture incubator. After 4 hours, add 10 μL of CCK-8 reagent to each well and continue incubating for another 2 hours. Measure the absorbance at 450 nm using a microplate reader and analyze the data. Results are as follows: Figure 6 As shown, there was no statistically significant difference in viability between CRISPR-NC cells transfected with cr5, cr10, cr14, cr31, and crRNA and wild-type BHK-21 cells. This indicates that the CRISPR / Cas13d system constructed from crRNAs corresponding to cr5, cr10, cr14, and cr31 did not affect the normal growth characteristics of BHK-21 cells; and that the constructed CRISPR / Cas13d transgenic cells have good safety.

Claims

1. crRNAs for targeting the foot-and-mouth disease virus genome, characterized in that, The crRNAs are selected from at least one of the target sequences shown in SQE ID NO.1-10.

2. The crRNA as described in claim 1, characterized in that, The crRNAs are selected from at least one of the target sequences shown in SQE ID NO.1-4.

3. The use of the crRNAs as described in claim 1 or 2 in the preparation of drugs or reagents for inhibiting foot-and-mouth disease virus.

4. A CRISPR / Cas13d system for inhibiting foot-and-mouth disease virus, characterized in that, The CRISPR / Cas13d system is obtained by ligating the crRNAs of claim 1 or 2 into an expression vector for the Cas13d protein.

5. The CRISPR / Cas13d system as described in claim 4, characterized in that, The expression vector for the Cas13d protein carries a nuclear localization signal.

6. The method for constructing the CRISPR / Cas13d system as described in claim 4, characterized in that, The method includes the following steps: (1) Add AAAA to the 5' end of the target RNA to obtain a reverse oligonucleotide; at the same time, obtain the corresponding DNA complementary strand according to the target RNA, and add AAAAC to its 5' end to obtain a forward oligonucleotide; synthesize the above forward oligonucleotide and reverse oligonucleotide respectively, and anneal the synthesized forward oligonucleotide and reverse oligonucleotide to form a double strand; (2) The double strands obtained in step (1) are ligated into the expression vector of the Cas13d protein to obtain the CRISPR / Cas13d system.

7. The method as described in claim 6, characterized in that, The expression vector for the Cas13d protein is an expression vector for the Cas13d protein carrying a nuclear localization signal.

8. A transgenic cell line, characterized in that, The cell line is obtained by transfecting host cells with the gene editing vector described in claim 5.

9. The use of the cell line as described in claim 8 in screening or preparing drugs that inhibit the proliferation of foot-and-mouth disease virus.

10. The application of the cell line as described in claim 8 in preparing an in vitro screening model for drugs against foot-and-mouth disease virus, or in preparing a cell model for studying the replication mechanism of foot-and-mouth disease virus.

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