To Explore Effects Of CRISPR/Cas13d System On Porcine Epidemic Diarrhea Virus Replication And Immune Status Of Vero Cells

Porcine epidemic diarrhea virus(PEDV),belonged to the genus Alphacoronavirus in the family Coronaviridae,is the pathogenic agent of porcine epidemic diarrhea(PED),which causes acute intestinal infectious diseases.The main symptoms of porcine epidemic diarrhea are acute diarrhea,vomiting and dehydration,and the mortality of piglets is very high,which has brought huge economic losses to the global pig industry.At present,gene editing technology has been widely used in plant and mammal gene editing research.Cas13,a member of the CRISPR protein family,can target RNA for degradation.In this project,N gene and Rd Rp gene of PEDV were selected as target genes.The smaller and precise CRISPR/Cas13 d system was used to degradate the target genes,and its effect on PEDV replication in Vero cells was determined.At the same time,the effect of CRISPR/Cas13 d system on the immune status of Vero cell was investigated by transcriptome.The main results were as follows:1.In vitro reporting system was used to study the effect of CRISPR/Cas13 system on PEDV gene expression.(1)Refer to current literatures,nucleocapsid protein N and RNA polymerase Rd Rp genes of PEDV were selected as candidate target genes.According to the N and Rd Rp sequences of different PEDV strains included in NCBI database,Clustal X2 software was used to conduct multiple sequence alignment.The conserved regions were selected and candidate cr RNA sequences with high scores were obtained on Cas13 d cr RNA Design website(https://Cas13 design.nygenome.org/).The cr RNA sequences were cloned into cr RNA scaffolds(PSLQ5429).In order to construct an in vitro recombinant reporter plasmid containing PEDV N and Rd Rp,the PEDV RNA was used as the template to amplify the full-length ORF of PEDV N and Rd Rp by PCR,and the ORF was cloned into p QCXIH-Mcitrine eukaryotic expression vector.Mooever,The cr RNA-N and cr RNA-Rd Rp sequences were cloned into to the All in One plasmid was constructed by ligation the cr RNA scaffold to the Cas13 d backbone.The results of gel electrophoresis and sequencing showed that the recombinant plasmids were all successfully constructed.(2)In order to explore the influence of cr RNA lengths on the gene inhibition effect,cr RNA with different length(22 bp,23 bp 28 bp and 30 bp)which targeted the same conservative region was designed.These 4 cr RNAs were co-expressed with Cas13 d and PQCXIH-Mcitrine-PEDV N in 293 T cells,respectively.24 h later,the PEDV N gene expression level was detected by quantitative PCR.The results showed that all lengths of cr RNA could inhibit the expression of PEDV N,and 22 bp cr RNA had a weak inhibition effect.It showed that the cr RNA of 23-30 bp could have a better inhibitory effect on the virus.(3)In order to screen out the best cr RNA and determine the most suitable conditions,cr RNAs targered different region were designed,and different doses of Cas13 d and cr RNA were applied.293 T cells were co-transfected with the constructed cr RNA knockout vector,Cas13 d plasmid and recombinant reporter plasmid,respectively.24 h later,fluorescence was observed under microscope,and the expression level of target genes was detected by quantitative PCR.The results showed that among the 4 pairs of cr RNAs targeting PEDV N gene,3 pairs(N1,N2 and N3)showed significant inhibitory effect.Among the 8 cr RNAs targeting PEDV Rd Rp gene,3 pairs of cr RNAs(R4,R5 and R7)had the best inhibitory effect.Different doses of Cas13 d have little effect on viral knockout effect,and high doses of cr RNA may damage cells and affect the inhibitory efficacy.2.Impact of CRISPR/Cas13 system on PEDV replication level(1)Plasmid 155305 containing CRISPR/Cas13 d system was transferred into Vero cells by packing lentivirus with the help of PMD2 G and PXPAX2 plasmids.After 48 h of infection,red fluorescence was observed under a fluorescence microscope,and cell clusters grown from single red fluorescence cells were screened by 2-fold dilution method.Then fluorescence quantitative PCR and gel electrophoresis were used to verify whether Cas13 d was integrated into the genome of Vero cells.The results showed that the expression level of Cas13 d in the lentivirus infected group was significantly higher than that in the control group,and the bands with the expected size could be obtained by gel electrophoresis,indicating the successful construction of Vero-Cas13 d stable cell lines.The results of CCK-8 test showed that Vero cells continued to grow within 72 h,while Vero-Cas13d-F4 and Vero-Cas13d-C5 cells had the same growth trend,and both reached the peak cell growth at 48 h,and Vero-Cas13d-F4 cells were relatively The fast growth rate of Vero-Cas13d-C5 indicated that Cas13 d could affect cell proliferation after being inserted into the cell genome.(2)Three cr RNAs(N3,R5 and R7)with the best inhibitory effect on target genes have been screened out in the preliminary experiment.These three cr RNAs were transfected into Vero-Cas13 d stable cell lines.24 h later,and PEDV infected the cells for another 24 h.Cell proliferation and PEDV N,S,M and Rd Rp gene expression levels were detected.The results showed that compared with the control group,the experimental group had more cells,and the expression levels of N,S,M and Rd Rp genes were significantly lower than those of the control group.Among them,cr RNA-R5 and cr RNA-R7 showed better virus inhibitory efficacy.The constructed All In One plasmid was overexpressed in Vero cells,24 h later,PEDV infected the cells for another 24 h.The expression levels of PEDV N,S,M and Rd Rp genes in the cells were detected.The results showed that the expression of viral genes in cells added with cr RNA was significantly reduced.The results of virus proliferation showed that the trend of virus proliferation in the cells added with cr RNA at 24 h was lower than that in the control group.The antiviral results of cr RNA showed that the cr RNA groups at different time points all showed the effect of inhibiting virus replication,and the results at 24 h were better.The above results indicate that the All in one plasmid constructed in this subject can inhibit the replication of PEDV.3.Effects of CRISPR/Cas13 system on Vero cell immunityIn order to explore whether the insertion of Cas13 d system into cells will changes the immune status of cells.Vero,Vero-Cas13-F4 and Vero,Vero-Cas13-C5 cells were subjected to high-throughput sequencing to analyze the differentially expressed genes in each group and perform GO enrichment analysis.Then some genes were selected for QRT-PCR verification.GO enrichment results showed that the functions of these DEGs were mainly involved in biological processes such as vascular function of circulatory system,formation of endoderm,transport of aromatic amino acids,positive regulation of epithelial cell differentiation,regulation of signal transduction mediated by small molecule GTASE,and formation of retinal layer.The above results show that the CRISPR/Cas13 d system can successfully degrade the target genes of PEDV and reduce the replication ability of the virus in cells.The CRISPR/Cas13 d system can affect the proliferation and immune status of cells,and the impact of this effect on cellular defense remains to be further studied.