Application of gRNA that inhibits porcine C16orf62 gene expression in the preparation of drugs against porcine delta coronavirus infection

Abstract

This invention discloses the application of gRNA that inhibits the expression of the porcine C16orf62 gene in the preparation of drugs against porcine delta coronavirus infection, belonging to the fields of genetic engineering and biomedicine. Based on CRISPR / Cas9 knockout technology, this invention constructs a porcine C16orf62 gene knockout cell line and finds that knocking out the porcine C16orf62 gene in LLC-PK1 cells can inhibit the proliferation of porcine delta coronavirus. This indicates that this gene can serve as a novel target for the development of vaccines and drugs that inhibit porcine delta coronavirus replication and for disease-resistant breeding research, providing new methods and directions for the prevention and control of porcine delta coronavirus and the diseases it causes.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biomedicine, and in particular to the application of gRNA that inhibits the expression of the porcine C16orf62 gene in the preparation of drugs against porcine delta coronavirus infection. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) is one of the pathogens causing severe diarrhea and death in piglets. This virus has posed a serious threat to the global pig industry and caused enormous economic losses. Although the successful isolation of PDCoV has greatly advanced the basic and applied research on this virus, no effective treatments or vaccines specifically targeting PDCoV infection have yet been developed, and the mechanisms of PDCoV infection remain poorly understood. Previous studies have shown that knocking out the PDCoV receptor gene ANPEP does not completely prevent viral infection, suggesting that other receptors or co-receptors are involved in the viral infection process. Understanding the infection mechanism of coronaviruses on their hosts is of great significance for the development of broad-spectrum antiviral drugs.

[0003] After binding to receptors on the cell surface, coronaviruses release their genome into the cytoplasm to initiate replication primarily through two pathways: First, the virus can directly invade the cell by fusing its envelope with the cell membrane; second, viral particles may also enter the cell via endocytosis, where the viral envelope fuses with the endosome membrane in early endosomes, late endosomes, or lysosomes, thereby releasing their genome for replication. When receptors on the cell surface bind to ligands derived from the cell or virus, endocytosis is initiated. The fate of the endocytosed receptors and complexes is determined by transport complexes such as the retrovesicle transport complex (also known as Retromer, composed of VPS26, VPS29, and VPS35). Some receptors and complexes are transported along microtubules via circulating endosomes, eventually returning to the plasma membrane, trans-Golgi apparatus, or viral replication region to perform their physiological functions. Others are transported through early endosomes to late endosomes or lysosomes, where they are eventually degraded or the viral genome is released. Retriever is a novel trimeric complex with a structure and function similar to the Retromer complex. It consists of C16orf62, VPS26, and VPS29, and is known as the heterologous retrograde vesicle transport complex. Both complexes are located in the same endosome domain and are associated with sorting linkers to select specific cargo, sorting transmembrane cargo back to the cell surface from the endosomal pathway. The role of Retromer in viral infection is relatively well understood; it can interact with various viral proteins or receptors, thereby influencing viral invasion, infection, and replication. Examples include hepatitis C virus (HCV), human papillomavirus (HPV), and SARS-CoV-2. However, the mechanism of action of Retriever in viral infection is relatively poorly understood.

[0004] To identify the receptor and infection-related host factors of PDCoV, a CRISPR / Cas9 high-throughput screening platform was established in PDCoV-susceptible Huh7 cells, and this technology was used to screen for the receptor and infection-related host factors of PDCoV. Using this platform, this invention discovered that the key gene C16orf62 (also known as VPS35L) of the Retriever complex was significantly enriched in the PDCoV-infected genome. Currently, there are no reports of C16orf62 participating in coronavirus infection. Summary of the Invention

[0005] The purpose of this invention is to provide the application of gRNA that inhibits the expression of the porcine C16orf62 gene in the preparation of drugs against porcine delta coronavirus infection, in order to solve the problems existing in the prior art. By knocking out the porcine C16orf62 gene in LLC-PK1 cells, it was found that porcine delta coronavirus proliferation can be inhibited, which provides a new strategy for the prevention and control of porcine delta coronavirus and the diseases it causes.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a gRNA that inhibits the expression of the porcine C16orf62 gene, the nucleotide sequence of which is: 5'-CAAAGAAAGTGAGCCGGAAG-3'.

[0008] The present invention also provides a knockout vector or knockout cell line containing the gRNA described above.

[0009] The present invention also provides a kit containing the gRNA described above.

[0010] Preferably, the kit also includes primer pairs for detecting porcine C16orf62 gene expression.

[0011] This invention also provides the use of the described gRNA, or the described knockout vector, or the described knockout cell line, or the described kit, in any of the following:

[0012] (1) Application in identifying the function of the porcine C16orf62 gene;

[0013] (2) Application in the preparation of drugs or cell models against porcine delta coronavirus infection;

[0014] (3) Application in the preparation of drugs that inhibit the proliferation of porcine delta coronavirus in LLC-PK1 cells.

[0015] The present invention also provides a method for constructing a mutant cell line that inhibits the expression of the porcine C16orf62 gene, comprising using the porcine C16orf62 gene as the target gene, knocking out the C16orf62 gene in the cell using a CRISPR / Cas9 gene editing system, and constructing a mutant cell line that inhibits the expression of the C16orf62 gene.

[0016] The gRNA sequence of the CRISPR / Cas9 gene editing system is 5'-CAAAGAAAGTGAGCCGGAAG-3', and the nucleotide sequence of the C16orf62 gene is the sequence shown in SEQ ID NO.1 or a gene sequence containing the sequence shown in SEQ ID NO.1.

[0017] Preferably, the cells comprise porcine cells.

[0018] Preferably, the porcine cells are porcine kidney cells.

[0019] The present invention also provides a mutant cell line that inhibits the expression of the porcine C16orf62 gene, which is constructed by the aforementioned construction method.

[0020] The present invention also provides the application of the mutant cell line described herein in screening for targets or drugs that inhibit the proliferation of porcine delta coronavirus.

[0021] The present invention discloses the following technical effects:

[0022] This invention utilizes CRISPR / Cas9 knockout technology to target and knock out C16orf62 in LLC-PK1 cells. It is the first discovery that cells with C16orf62 knockout can resist PDCoV infection by inhibiting PDCoV proliferation. This gene can serve as a novel anti-PDCoV target. This invention provides a new target for the development of effective and safe novel vaccines and drugs to prevent and control PDCoV, and also provides a new candidate target gene for breeding research against PDCoV, demonstrating broad application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 To design gRNAs targeting the porcine C16orf62 gene;

[0025] Figure 2 To construct a knockout plasmid map targeting the porcine C16orf62 gene;

[0026] Figure 3 Electrophoresis diagram for PCR identification of plasmid construction in bacterial culture; M is Trans 2K Plus DNA Marker, 1-5 are sgLenti-C16orf62 KO sgRNA bacterial cultures, and 6 is the negative control;

[0027] Figure 4 The results were used to confirm the successful construction of the plasmid through sequencing.

[0028] Figure 5 Western blot analysis of the successful construction of the LLC-PK1 Cas9 cell line.

[0029] Figure 6 The results of the construction and validation of the LLC-PK1 Cas9-C16orf62 KO monoclonal cell line; (A) Gene DNA sequencing of C16orf62 gene sequence in NC KO and C16orf62 KO cells; (B) CCK-8 assay for the viability of NC KO and C16orf62 KO cells;

[0030] Figure 7 The effect of C16orf62 knockout on PDCoV infection was observed using fluorescence microscopy after DAPI staining.

[0031] Figure 8 To analyze the impact of C16orf62 knockout on PDCoV infection using flow cytometry;

[0032] Figure 9 To determine the proportion of cells infected with PDCoV after C16orf62 knockout (*P<0.05, **P<0.01, ***P<0.01, t-test);

[0033] Figure 10 To analyze the impact of C16orf62 knockout on PDCoV infection using qRT-PCR;

[0034] Figure 11 The growth curves of PDCoV in NC KO and C16orf62 KO cells are shown. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Example 1: Construction and Validation of C16orf62 Knockout Cell Line

[0041] The NCBI number of the mRNA of the target gene C16orf62 is NC_010445.4, and the corresponding CDS sequence is shown in SEQ ID NO.1, encoding the protein shown in SEQ ID NO.2.

[0042] CDS sequence:

[0043]

[0044] amino acid sequence:

[0045] 。

[0046] 1. gRNA design and expression vector construction

[0047] (1) gRNA design: Select a partial sequence of the gene location to which the gRNA needs to target, and design the knockout gRNA sequence (see below). Figure 1 ; gRNA: 5'-CAAAGAAAGTGAGCCGGAAG-3' (SEQ ID NO. 5).

[0048] (2) Anneal the single-stranded gRNA synthesized by the company to double-stranded gRNA. The annealing system is as follows: 10 μL each of upstream and downstream sgRNA, 2.5 μL of ddH2O, 2.5 μL of T4 Buffer, and 1 μL of polynucleotide 5' hydroxykinase (PNK). The program is as follows: 37℃ for 30 min, 95℃ for 5 min, 80℃ for 5 min, 75℃ for 5 min, 70℃ for 5 min, and 20℃ for 5 min.

[0049] (3) The sgLenti(MP-783)(Tromp, et al., 2018, “Human CD45 is an F-component-specific receptor for the staphylococcal toxin Panton-Valentine leukocidin”) vector was digested and recovered using Aar I enzyme, and the gRNA was ligated to the vector using T4 ligase to construct the knockout vector.

[0050] (4) The vector was transformed into DH5α, plated, and single colonies were picked for PCR amplification using 2×EsTaqMasterMix(Dye) enzyme from Kangwei Century Company. The amplification primers were: F: 5'-ATAACTTCGTATAGCATACATTAT-3'; R: 5'-GCACATGCAGTGACTGGAGT-3'. The PCR system was set as follows: 12.5 μL of 2×EsTaq MasterMix(Dye), 1 μL each of forward and reverse primers, 2 μL of template, and 8.5 μL of ddH2O. The program was as follows: pre-denaturation at 94℃ for 2 min, followed by cycling the reaction as follows: denaturation at 94℃ for 30 sec, annealing at 56℃ for 30 sec, extension at 72℃ for 30 sec, for a total of 35 cycles, and a final extension at 72℃ for 2 min. The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0051] (5) Positive colonies verified by PCR and sequencing (see...) Figure 3 and Figure 4Following the instructions of the TIANGEN Plasmid Mini-Prep Kit (DP103), plasmids were extracted from the amplified bacterial culture. The resulting plasmid was named sgLenti-C16orf62 KO vector (see figure). Figure 2 ).

[0052] like Figures 1-4 As shown in the figure, the results indicate that the gRNA targeting the C16orf62 gene was successfully ligated into the plasmid, indicating that the gRNA expression vector was successfully constructed.

[0053] 2. Construction of LLC-PK1 Cas9 cell line

[0054] Following the instructions for use of the JetPRIME DNA transfection reagent (114-15), lentivirus pLenti-Cas9-Blast (Addgene, 52962) was packaged using helper plasmids psPax2 (Addgene, 12260) and pMD2.G (Addgene, 12259).

[0055] LLC-PK1 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 24-well cell culture plates and divided into transduction and control groups. When the cell confluence reached 40%–50%, pLenti-Cas9-Blast lentivirus (MOI = 1) was diluted in DMEM high-glucose medium containing 10% FBS, 1% penicillin-streptomycin, and 8 μg / mL polybrene and transduced into LLC-PK1 cells. After 1 day, the medium was replaced with fresh complete medium for further culture. After 2 days, the medium was replaced with complete medium containing 6 μg / mL blastcinin (Blast) for selection. After all cells in the control group died, the surviving cells in the experimental group were serially diluted and seeded into 96-well cell plates to form single cells. The cells were then cultured in Blast medium until they grew into monoclonal cell clusters. These clusters were then digested with trypsin and expanded in 24-well culture plates to obtain the monoclonal cell line LLC-PK1 Cas9. Western blot analysis was performed to detect Cas9 expression.

[0056] (1) Preparation of protein samples: LLC-PK1 cells and LLC-PK1 Cas9 cell lines were evenly seeded into 6-well plates. After the cells were fully grown into a monolayer, the culture medium was discarded and the cells were washed twice with pre-cooled PBS.

[0057] (2) Add lysis buffer containing PMSF and protease inhibitor (abcam, ab141032) to each well, lyse on ice for 15 min, scrape cells with a cell scraper and aspirate the lysate into a 1.5 mL centrifuge tube, repeatedly pipette with a 1 mL syringe until clear, add 5×SDS-PAGE loading buffer, boil in a 95℃ metal bath for 10 min, and wait for sample loading and detection.

[0058] (3) Perform SDS-PAGE electrophoresis.

[0059] (4) Perform Western Blot analysis:

[0060] a) The primary antibody was Cas9 Rabbit pAb (ABclonal, A14997), and the secondary antibody was HRP-conjugated Goatanti-Rabbit IgG (H+L) (ABclonal, AS014).

[0061] b) Protein detection: ECL luminescence method was used.

[0062] The results are as follows Figure 5 As shown, Western blotting revealed that LLC-PK1 Cas9 cells expressed Cas9 protein, while the control group LLC-PK1 cell line did not express Cas9 protein, indicating that LLC-PK1 Cas9 cells were successfully constructed.

[0063] 3. Constructing a C16orf62 knockout monoclonal cell line

[0064] (1) Lentiviral gRNA was packaged using helper plasmids psPax2 (Addgene, 12260) and pMD2.G (Addgene, 12259) according to the instructions of the DNA transfection reagent (114-15) from JetPRIME.

[0065] (2) LLC-PK1 Cas9 cells were seeded into 24-well cell culture plates and divided into transduction group and control group. They were cultured in DMEM high glucose medium containing 10% FBS and 1% penicillin-streptomycin.

[0066] (3) When the cell confluence reaches 40% to 50%, dilute the gRNA lentivirus targeting gene C16orf62 (MOI=1) in DMEM high glucose medium containing 10% FBS, 1% penicillin-streptomycin and 8 μg / mL polybrene, and transduce it into LLC-PK1 Cas9 cells.

[0067] (4) One day after infection, replace with fresh complete culture medium and continue culturing.

[0068] (5) Two days after infection, the medium was replaced with a complete medium containing 4 μg / mL puromycin (10% FBS, 1% penicillin-streptomycin DMEM high glucose medium).

[0069] (6) Replace the fresh complete culture medium containing puromycin every day. When all the cells in the control group have died and some cells in the experimental group have survived, the cells in the experimental group are serially diluted and seeded into 96-well cell plates to form single cells. Continue to culture them in culture medium containing puromycin.

[0070] (7) Continue culturing for about 7 days until the individual cells grow into monoclonal cell clusters. Select monoclonal cells, digest them with trypsin, and place them in a 24-well culture plate to obtain a monoclonal cell line (C16orf62 KO).

[0071] 4. Verify the expression of the C16orf62 gene and cell viability in the C16orf62 KO cell line.

[0072] 4.1 Genomic DNA Sequencing Detection of C16orf62 Gene DNA Editing in C16orf62 KO Monoclonal Cells

[0073] (1) Extraction of cellular genomic DNA

[0074] DNA was extracted from the C16orf62 knockout monoclonal cell line and the control group LLC-PK1 Cas9 cells (NC KO cells) according to the instructions of the Tiangen DNA Extraction Kit (DP304).

[0075] (2) To verify the sequence near the C16orf62 gene knockout site, PCR amplification was performed using PrimeSTARMax DNA Polymerase (R045Q) provided by Takara, and the sample was then sent for sequencing. The amplification primers were:

[0076] C16orf62 KO-F: 5'-AGTGGCCGTGTCGCTCCCTT-3';

[0077] C16orf62 KO-R: 5'-GGAAGCAGCAGCAGTTTCCAGGATCTA-3'.

[0078] The PCR program was set as follows: pre-denaturation at 98℃ for 15 seconds, followed by cycling of denaturation at 98℃ for 10 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 1 min, for a total of 35 cycles, and a final extension at 72℃ for 5 min. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0079] Sequencing results as follows Figure 6As shown in Figure A, it was found that the C16orf62 gene frameshift mutation was caused by the deletion of 7 base pairs in the C16orf62 KO monoclonal cell line, resulting in the non-production of C16orf62 protein.

[0080] The amplified sequence of NC KO cells before knockout is shown in SEQ ID NO.3; the amplified sequence of the C16orf62 KO monoclonal cell line after knockout is shown in SEQ ID NO.4.

[0081] SEQ ID NO.3 is:

[0082] .

[0083] SEQ ID NO.4 is:

[0084] .

[0085] 4.2 Detection of C16orf62 KO monoclonal cell line viability using CCK-8 assay

[0086] (1) Cell viability was detected according to the CCK-8 kit (Beyotime, C0037) instructions. NC KO cells and C16orf62 KO cells were seeded into 96-well plates with 3 replicates per group and cultured in a 5% CO2, 37°C incubator for 48 h.

[0087] (2) Take out the 96-well plate, add 10 μL of CCK-8 solution to each well, and continue to incubate in the cell culture incubator for 4 hours.

[0088] (3) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 450 nm.

[0089] The results of the CCK-8 experiment are as follows: Figure 6 As shown in Figure B, the results indicate that there was no difference in cell viability before and after C16orf62 knockout, suggesting that knocking out the C16orf62 gene had no significant effect on cell viability.

[0090] Example 2: Detection of the effect of C16orf62 gene knockout in LLC-PK1 cells on PDCoV infection.

[0091] The inhibitory effect of C16orf62 gene knockout on PDCoV infection in LLC-PK1 cells was detected using fluorescence microscopy, flow cytometry, and qRT-PCR. The specific procedures are as follows:

[0092] C16orf62 KO cells and control NC KO cells were seeded into 24-well plates and cultured in a 5% CO2 incubator at 37°C. After 24 hours, the culture medium was discarded, and the cells were washed three times with PBS. The medium was then replaced with PDCoV-GFP virus solution diluted in DMEM medium containing 10 μg / mL trypsin (MOI = 0.1). This virus was constructed and preserved in our laboratory (refer to Zhang et al., 2020, “Genetic manipulation of porcine deltacoronavirus reveals insights into NS6 and NS7 functions: anovel strategy for vaccine design”). After incubation at 37°C for 1 hour, the cells were washed three times with PBS, and then cultured again with medium containing 10 μg / mL trypsin. The supernatant was collected at 12 hours and 24 hours after infection. The cells were observed under a fluorescence microscope and analyzed using a cytometer. The virus solution collected at 24 hours was identified by qRT-PCR.

[0093] (1) The effect of C16orf62 knockout on PDCoV infection was observed by DAPI staining and fluorescence microscopy.

[0094] (2) Flow cytometry analysis was used to detect the effect of C16orf62 knockout on PDCoV infection.

[0095] (3) For quantitative real-time detection, viral RNA was first extracted using the Tiangen Virus RNA Extraction Kit (YDP315-R), and cDNA was generated using the Novozymes HiScript II 1st Strand cDNA Synthesis Kit (R211-01) as a template. Amplification was performed using the Takara Probe Detection Kit (RR600B) on a Bio-Rad CFX96 instrument. The standard plasmid was constructed and preserved in the laboratory, with the probe sequence 5'-CACACCAGTCGTTAAGCATGGCAAGCT-3', and the upstream and downstream primer sequences were F: 5'-ATCGACCACATGGCTCCAA-3', R: 5'-CAGCTCTTGCCCATGTAGCTT-3'. The reaction system and procedure were performed according to the manufacturer's instructions.

[0096] The results are as follows Figures 7-10 As shown, fluorescence microscopy and flow cytometry analysis revealed that, 12 h and 24 h after infection, the number of PDCoV-infected cells in C16orf62 KO cells was significantly lower than that in the NC KO control group. qRT-PCR results further demonstrated a decrease in PDCoV RNA copy number after C16orf62 knockout, indicating that knocking out the C16orf62 gene in LLC-PK1 cells significantly inhibited PDCoV proliferation.

[0097] Example 3

[0098] Plot a one-step growth curve using TCID 50 The absence of the C16orf62 gene in LLC-PK1 cells was found to inhibit the proliferation of PDCoV.

[0099] C16orf62 KO cells and control NC KO cells were seeded into 24-well plates and cultured at 37°C with 5% CO2. After 24 hours, the original culture medium was discarded, and the cells were washed three times with PBS. The medium was then replaced with PDCoV-GFP diluted in DMEM containing 10 μg / mL trypsin. After incubation at 37°C for 1 hour, the cells were washed three times with PBS, and then cultured again with medium containing 10 μg / mL trypsin. The supernatant was collected at 6, 12, 24, 36, and 48 hours post-infection for PDCoV titer detection. The experiment was repeated three times. The specific detection method is as follows.

[0100] (1) Seed LLC-PK1 cells into 96-well plates and wait for them to grow into a monolayer for later use.

[0101] (2) The collected virus solution was serially diluted 10-fold with DMEM containing 10 μg / mL trypsin, with the dilution range starting from 10 μg / mL. -1 Up to 10 -10 .

[0102] (3) Add 100 μL of diluted virus solution to each well, and set up 8 replicate wells for each dilution. At the same time, use DMEM without virus as a blank control.

[0103] (4) Place the 96-well plate in a 37°C, 5% CO2 incubator and observe it daily. After 2-3 days, count the number of wells infected with PDCoV.

[0104] (5) Calculated using the Reed-Muench method, specifically as follows: Distance Ratio = (Percentage of lesions above 50% - 50%) / (Percentage of lesions above 50% - Percentage of lesions below 50%), lgTCID 50= Distance ratio × Difference between the logarithms of dilutions + Logarithm of dilutions with a lesion rate higher than 50%.

[0105] The results are as follows Figure 11 As shown in the one-step growth curve, it can be observed that knocking out C16orf62 in LLC-PK1 cells significantly reduced the PDCoV titer, indicating that knocking out this gene in LLC-PK1 cells can inhibit the proliferation of PDCoV.

[0106] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of gRNA to inhibit porcine C16orf62 gene expression: (1) Application in the preparation of drugs or cell models against porcine delta coronavirus infection; (2) Application in the preparation of drugs that inhibit the proliferation of porcine delta coronavirus in LLC-PK1 cells; The nucleotide sequence of the gRNA is: 5'-CAAAGAAAGTGAGCCGGAAG-3'.

2. Application of the knockout vector or knockout cell line containing the gRNA described in claim 1: (1) Application in the preparation of drugs or cell models against porcine delta coronavirus infection; (2) Application in the preparation of drugs that inhibit the proliferation of porcine δ-coronavirus in LLC-PK1 cells.

3. Application of the kit containing the gRNA described in claim 1: (1) Application in the preparation of drugs or cell models against porcine delta coronavirus infection; (2) Application in the preparation of drugs that inhibit the proliferation of porcine δ-coronavirus in LLC-PK1 cells.

4. The application as described in claim 3, characterized in that, The kit also includes primer pairs for detecting porcine C16orf62 gene expression.

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