Study On Genomic Edition Of Porcine Mx1 Gene Based On CRISPR/Cas9 Technique

Porcine circovirus type 2(PCV2)is a virus that causes congential tremors and postweaning multisystemic wasting syndrome,resulting in huge economic losses to the pig industry.How to reduce the loss caused by PCV2 to the pig industry becomes more and more important.In recent years,the applications of the CRISPR/Cas9 system to genetic breeding and gene therapy are in progress,making the use of the CRISPR/Cas9 system to produce PCV2-resistant pigs more important to the pig industry.Mx1 protein,one of the interferon(IFN)induced proteins,has activity against a variety of viruses.In our previous study,the 275 bp insertion in the promoter region of the Laiwu pig Mx1 gene at-547 bp is related to the insensitivity of Laiwu pigs to PCV2.In this study,we performed a site-directed insertion of 275 bp in the promoter region of the Mx1 gene in PK15 cells and verified its ability to stimulate Mx1 expression and inhibit PCV2 replication by using CRISPR/Cas 9 and Cre-Lox P systems.The results of this study were shown below.(1)Construction of porcine Mx1 gene CRISPR/Cas9 homologous recombination vector and targeting vector.Using the PLV-m Cherry as the backbone,we designed and obtained the homology arm based on the difference of 275 bp insertion site in the promoter region of pig Mx1 gene,and inserted green fluorescent protein and puromycin resistance genes and added Lox P sequences in the same direction at both ends,and finally obtained CRISPR/Cas9 homologous recombination vector.We designed sg RNAs for Mx1 and inserted into Lenti CRISPR v2 vector according to the instruction book.Then the digestion efficiency was identified by the T7E1 enzyme.(2)Identification of homologous recombination monoclonal cells in porcine PK-15 cells.PK-15 target cells were infected with packaged virus(containing Lenti-CRISPR and homologous recombination vectors),and selected by Puromycin.Two monoclonal cells were obtained and identified by PCR(polymerase chain reaction).(3)Removal of marker genes.Monoclonal cells were transfected with a vector expressing Cre enzyme.By using the fluorescence microscope,we found that the luminous cells were significantly reduced.And flow cytometry analysis proved that the proportion of green fluorescence was reduced(16.3%).It is proved that the Cre-Lox P system removed the selectable marker gene in the cell by cutting in the same direction.(4)Expression of monoclonal cell Mx1 and inhibition of PCV2 replication in PK-15 cell.Gene-edited monoclonal cells were obtained by insertion of the 275 bp site in Mx1 promoter region and inoculated with PCV2.The expression level of Mx1 was significantly increased at 48h after PCV2 inoculation,and the PCV2 DNA copy was significantly reduced.It shows that the insertion of 275 bp in the promoter region of pig Mx1 has the function of promoting Mx1 expression and inhibiting PCV2 replication.