CRISPR/Cas9 Mediated HFAD3 Gene Knock-In At NCAPG-LCORL Locus In Bovine

The present study was aimed to knock-in the marker-free hFAD3 gene expression vectors at NCAPG-LCORL locus mediated by the CRISPR/Cas9 in bovine, to study the fatty acids expressions in the transgenic cells and the expressions of the flanking genes and fatty acids associated genes, and to analyze the knock-in efficiency and hFAD3 expressions with the involvement of the MAR element.The results showed that:(1) Five hFAD3 gene targeting vectors at the NCAPG-LCORL locus were successfully constructed, they were C2 (5’arm-CAG-hFAD3-PolyA-3’arm), M2 (5’arm-MAR-CAG-hFAD3-PolyA-MAR-3’arm), P2 (pGT-C1-LHA-RHA-hFAD3), C22 (LHA-CAG-hFAD3-PolyA-3’arm) and CRL (LHA-CAG-hFAD3-PolyA-RHA). (2) All of these vectors were successfully knocked-in at NCAPG-LCORL locus in the bovine cell genomes. (3) The insertion of the foreign genes affected the expressions of the flanking genes. (4) In transgenic cells, the fatty acids composition were changed as decreased the ω6 PUFAs and the ration between ω6 and ω3 significantly decreased (P<0.01). (5) In the transgenic cells, the fatty acids metabolism associated genes expressed changed to tend to lipolysis other than synthesis. (6) Out of 59 monoclones 3 strains homologous recombinant monoclones were obtained by flow cytometry and infinite dilution culture and PCR identification. The konck-in efficiency was 5.1%. The expressions of fatty acids and flanking genes and fatty acids related genes in the monoclonal cells were also detected and the results were in consistent with the routinely transgenic cells. (7) The present of MAR significantly increased hFAD3 expression in the transgenic cells as compared to that without MAR (P<0.01).In conclusion, the hFAD3 gene could successfully knock-in at NCAPG-LCORL locus mediated by the CRISPR/Cas9 and normally expressed at the transgenic cells. The involvement of MAR element significantly increased the hFAD3 expression. This study may provide an alternative protocol to construct transgenic vector and probably useful to produce transgenic animals.