Generation Of TGEV-resistant Pigs With APN Seventh Structural Domain Gene Editing

Transmissible gastroenteritis(TGE)is an acute,highly contact infectious intestinal disease caused by transmissible gastroenteritis virus(TGEV).The symptoms are mainly vomiting,diarrhea and dehydration,and the fatality rate can reach 100% in piglets within2 weeks of age.Porcine transmissible gastroenteritis is cyclically prevalent worldwide,causing serious losses to the pig industry.TGEV belongs to α coronavirus,it is a single-stranded positive-stranded RNA,which mainly includes three structural proteins: S protein,N protein and M protein.The amino terminal of the S protein is a closely related area where TGEV recognizes target cells,induces neutralizing antibodies and determines the tropism of virus tissue.The main receptor of TGEV is pig aminopeptidase N(APN).In recent years,a number of studies have shown that APN knockout pigs can significantly improve the ability to resist TGEV infection.However,in view of the important physiological functions of APN in terms of immunity,metabolism,cell movement and adhesion,this study proposes to exogene the key structural domain of APN in virus binding in order to avoid greater impact on the health and production performance of pigs.The porcine APN domain VII(717-839 amino acids)gene sequence was replaced with homologous murine APN domain VII sequence to obtain gene-edited pigs.Replacing the key structural domain of TGEV binding with murine genes is promising to improve the resistance of pigs to TGEV while retaining the other important physiological functions of APN to the greatest extent.In this study,through CRISPR/Cas9-mediated homologous recombination technology,a homologous replacement of the murine APN domain VII sequence was achieved at the gene sequence site of the pig genome APN domain VII(amino acids717-839)and was safe and effective expression.After evaluating the anti-TGEV effect of positive clones at the cellular level,APN gene-edited pigs were successfully prepared.First,we constructed the sg RNA targeting vector and the APN mouse-derived replacement plasmid Donor,which were co-transfected into PK-15 cells by electrotransfection.After 48 hours,they were plated and screened and identified by limiting dilution.Confirmed by agarose gel electrophoresis and Sanger sequencing,positive PK-15 cells were obtained.we inoculated the PK-15 positive cell clones and wild-type PK-15 with TGEV,extracted total RNA,and detected the difference in virus copy number between the two groups by RT-PCR;extracted protein,and detected at the protein level by Western Blotting.The proliferation of the virus in the two groups was used to evaluate the anti-TGEV effect of positive cell clones.The results of inoculation at the cell level showed that the virus proliferation of the two groups of cells was significantly different,and the positive cell clones could significantly resist TGEV infection.Porcine fetal fibroblast cell clones were selected in the same way.We use the positive fibroblast clones as donor cells,and use SCNT technology to prepare anti-TGEV gene-edited pigs to obtain F0 generation positive clone pigs and then multiply to obtain F1 generation positive pigs.The kidney cells of F1 positive pigs and wild-type pigs were isolated and cultured,and TGEV was inoculated.The differences between the two groups were assessed by RT-PCR and Western Blotting.Results showed that the kidney cells isolated from gene-edited pigs had significant antiviral properties.Taken together,these results suggested that CRISPR/Cas9-mediated knock-in strategy can be used for producing APN gene-edited pigs,which promised to be used to protect from TGEV.