Preparation Of Anti-CSFV/PEDV Genetically Modified Pigs Based On Porcine MiR-17-92 Cluster

Due to its high editing efficiency and rapid construction,CRISPR/Cas9 technology has greatly promoted the research of site-specific integration of foreign genes.The efficient knock-in of a foreign gene requires a safe site to allow its integration and efficient expression without affecting the intrinsic gene function in the cell or organism.Currently,two common sites for the integration of foreign genes in the pig genome are the Rosa26 site and the H11 site.However,the safe integration sites for transgenic research and preparation of genetically modified animals are far from enough.Therefore,this requires us to find more suitable sites for integration of foreign genes in the pig genome.Based on the porcine miR-17-92(pmiR-17-92)gene cluster and the CRISPR/ Cas9-mediated HDR pathway,this study successfully demonstrated the integration of exogenous genes and the potential of genetically modified pigs in the pmiR-17-92 cluster.First,we successfully constructed an EGFP reporter cell line using CRISPR/Cas9 technology.Through CRISPR/Cas9-mediated site-directed integration,we successfully integrated the shRNA that specifically inhibits the EGFP gene into the 3' UTR of pmiR-17-92.The shRNA gene inserted under the control of pmiR-17-92 was normally transcribed and effectively inhibited the expression of EGFP.This indicates that the pmiR-17-92 allows integration and efficient expression of foreign genes.On the basis of the above,we obtained a porcine fetal fibroblast cell line integrated with anti-CSFV shRNA by screening.We found that the anti-CSFV shRNA gene not only efficiently expressed after the insertion at 3' UTR of pmiR-17-92,but also did not affect the cell proliferation ability and early embryo development.We also analyzed in depth the integrity of the siRNA sequence generated by transcription in this cell line.By sequencing and sequence alignment,we found that the target siRNA sequence was completely correct.In addition,we did not find off-target effects at the 16 potential off-target sites we detected.Subsequently,we used the above cell line as a donor cell to prepare the anti-CSFV gene-editing pigs through SCNT.However,no successful pregnant sow was found during the preparation of genetically modified pigs.It is speculated that this is due to the small number of surrogate sows used for embryo transfer.In addition,we screened the sgRNA sequence that efficiently target human miR-17-92(hmiR-17-92)and successfully demonstrated the potential of hmiR-17-92 to integrate foreign genes in human HEK 293 cells.Finally,we successfully obtained an integrated cell line carrying the shRNA gene against PEDV in pmiR-17-92 and prepared related genetically modified pigs.We found normal transcription and efficient expression of the shRNA gene against PEDV in positive piglets,and it did not affect endogenous expression of pmiR-17-92.In addition,we examined potential off-target effects and found no off-target mutations in these genetically modified pigs.This study demonstrates that exogenous genes inserted into the pmiR-17-92 cluster are highly expressed in cell and individual levels.In summary,the site-specific integration and expression strategy of exogenous shRNA gene in porcine miR-17-92 cluster in this study laid the foundation for future antiviral research and genetically modified pig preparation.