| Porcine epidemic diarrhea virus(PEDV)is one of the most important enteric coronavirus causing diarrhea in piglets.However,the key receptor genes of PEDV have not been clarified and the pig breeds resistant to the virus cannot be fundamentally prepared.Clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9(CRISPR/Cas9)has been widely confirmed as an efficient and powerful third-generation gene editing tool.CRISPR/Cas9 gene editing system has been successfully applied in the screening of viral key genes in combination with genome-wide screening and subsequent functional gene identification,but it has not yet been applied on PEDV.Therefore,in this study,CRISPR high-throughput screening strategy was used to explore PEDV replication key candidate genes,which is expected to achieve the preparation of gene-edited pigs and provide a new direction for disease resistance molecular breeding research in pigs.Objective: In this study,genome-wide CRISPR/Cas9 technology was used to screen PEDV replicationrelated genes,and the candidate gene MMP13 was mechanistically studied on IPEC-J2 cells,which is a kind of pig-derived targeted cells,to provide a scientific reference for pigs resistant to PEDV.Methods: A genome-wide knockout library of human hepatocellular carcinoma cell line(Huh-7)was constructed by CRISPR/Cas9 technology.Then this library was infected with PEDV,and the key genes of PEDV replication were mined by high-throughput sequencing.Finally,the intrinsic mechanism of PEDV regulation by candidate gene MMP13 was explored in combination with molecular biological methods.Results:(1)In this study,we constructed a genome-wide screening method for PEDV replication-related genes by the CRISPR/Cas9 system,and the GO analysis results of the enriched target genes showed that these were mainly involved in biological processes such as double strand break repair,negative regulation of mitotic cell cycle and programmed cell apoptosis;KEGG analysis showed that these target genes were mainly involved in signaling pathways such as apoptosis,tumor transcription regulation,mitophagy,and cell cycle;RNA interference fragments were synthesized for the enriched top-ranked integrin α11(ITGA11),mammalian replication protein A2(RPA2),kinesin family member 2A(KIF2A),induced myeloid leukemia cell differentiation protein 1(MCL1),poly ADP-ribose 1(PARP1),and vesicular monoamine transporter 1(SLC18A1)genes,respectively,and subsequently transfected into IPEC-J2 cells.Compared with the control group,the interference with ITGA11 gene significantly decreased PEDV m RNA(P<0.01),protein expression levels and progeny virus titers;In addition,PEDV infection can significantly up-regulate the expression level of MMP13 gene,and has a high similarity with PEDV N protein expression trend.(2)In this study,we synthesized the interfering fragment of MMP13 gene and its overexpression vector.It was found that interference with MMP13 gene could significantly reduce the PEDV N m RNA(P<0.001)and protein expression levels on IPEC-J2 cells,while PEDV expression was significantly enhanced after overexpression of MMP13 gene.In addition,the protein expression level of PEDV N was also reduced after using MMP13-specific inhibitors,indicating that MMP13 gene can be used as a key gene to regulate the replication of PEDV.(3)Firstly,we measured the PI3K/AKT and JNK phosphorylated protein expression after PEDV infection in IPEC-J2 cells.Western blot results showed that PEDV infection could activate PI3K/AKT and JNK protein phosphorylation.Subsequently,PEDV AH2012/12 strains were infected after treatment with PI3 K phosphorylation inhibitor LY294002 and phosphorylation inhibitor SP600125 of JNK protein,respectively.The results showed that the expression levels of P-JNK,MMP13 and PEDV N protein were significantly decreased after treatment with SP600125,while the expression levels of MMP13 and PEDV N protein were not significantly changed after treatment with LY294002,indicating that PEDV mainly upregulated the expression of MMP13 through JNK signaling pathway;Moreover,confocal laser scanning assay and RT-q PCR results showed that the adsorption of PEDV to cells was extremely significantly decreased after interfering with MMP13 gene(P<0.001),and there was no change in invasion,replication and release stage(P>0.05);Conversely,the adsorption of PEDV to host cells was significantly increased after overexpression of MMP13 gene(P<0.05),indicating that MMP13 played a key role in the PEDV adsorption stage;Finally,confocal laser scanning microscopy assay revealed that MMP13 co-localized with Spike proteins.Conclusion: The genome-wide knockout library based on CRISPR/Cas9 system can be used as an effective tool to screen PEDV replication-related functional genes,and ITGA11 and MMP13 genes can be used as a potential target gene against PEDV,so as to fundamentally develop PEDV-resistant breeds or strains of pigs through disease resistance breeding approaches. |
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