CRISPR/Cas9-based Generation Of Anti-CSFV Pigs

Classical swine fever [CSF] caused by classical swine fever virus [CSFV] isamong the most detrimental diseases,which leads to significant economic losses in the swine industry.Despite efforts by many government authorities to stamp out the disease from national pig populations,the disease remains widespreadin many countries.These highlight thenecessity and the urgency to develop more effective approaches to eradicate CSFV.In this study,fisrt,we optimized and improved the CRISPR/Cas9-based knock-in efficiency in PFFs.Then weconfirmed that the combined application of CRISPR/Cas9 and RNAi,resulting in the generation of anti-CSFV TG pigs.More importantly,the in vitro and in vivo CSFV challenge assay demonstrated that these TG pigs exhibited higher resistance to CSFV infection than non-transgenic pigs.In addition,we also investigated precise point mutation of LamR on CSFV infection and successfully producted precise base editing Pigs via a CRISPR/Cas9-based point mutation technology.In summary,this study confirmed that the ability of pigs to resist viruses can be enhanced through genetic engineering,and it may provide a reference for disease-resistance breeding and future antiviral research in the future.Through research,this study mainly achieves results as follows:1.We further improved the electroporation efficiency in PFFs and confirmed that this electroporation system would also obtain high transfection efficiencies in various cell lines.Afteroptimization,the electroporation efficiency of the PFFs reachedapproximately 96.45%.Compared with other sgRNAs,sgRNA91 could achieve a higher targeting/cutting efficiency at pRosa26 locus.Additionally,we found that 30?g of the sgRNA91/Cas9 vectors could achieve a highertargeting/cuttingefficiency [34.3%] with less cytotoxicity.2.To achieve a safer transgenic strategy,a non-linearized targeting donor vector that did not contain a promoter or a selectable marker was used in this study.We achieved site-specific EGFP insertion at the SgRNA91 locus and confirmed this locus could be used to express EGFP,effectively.After optimization,the efficiency ofthe EGFP site-specific knock-in reached up to approximately29.6%.Additionally,we used the EGFP reporter PFFs to address two key conditions in the process of achieving transgenic pigs,the limiting dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus.This study may provide insights into sophisticated site-specific genome engineering techniques for additional species.3.Antiviral small hairpin RNAs[shRNAs] was selected and shRNA knock-in cell lines were selected by limiting dilution method.Finally,anti-CSFV transgenic [TG] pigs were produced by somatic nucleartransfer [SCNT].Importantly,in vitro and in vivo viral challenge assay demonstrated that these TG pigs exhibited a higher resistance to CSFV infection than non-transgenic [NTG] pigs,and the disease resistance traitscould stably transmit to F1-generations.In addition,there wasno selectable marker genes were introduced during the production of TG pigs,so as to reduce potential risk of drug selection and increase biological safety of TG pigs.4.LamR precise point mutation cell lines were selected by CRISPR/Cas9-based precise point mutation technology and viral challengeresultsconfirmed these point mutation cell lines enhanced resistancesto CSFV infection.Finally,the precise point mutation pigs were produced by somatic nucleartransfer.Importantly,in vitro viral challenge assay demonstrated that these TG pigs exhibited a higher resistance to CSFV infection.Because these antiviral pigs were edited via CRISPR/Cas9-based precise point mutation technology,they do not contain any transgenes,it will help to increase biological safety and provide market support.Thisstudymaycontributetothebetterunderstandingof viral attachment and entry and can help to enhance our understanding of the infection cycles of CSFV.Additionlly,this study can providetargets and therapeutic potential in viral diseases.