The Integration Of Multi-gene In Buffalian Cells Caused By CRISPR/Cas9

In large animals,most traits are quantitative traits,and buffalo's milk production is a complex quantitative trait.In most of the previous studies,it was difficult to control the production of a target gene,and it was difficult to achieve the purpose of regulating milk production and milk quality,and most of the studies were on the knockout of the gene,and the study less.At present,transgenic technology in the multi-gene joint regulation is not mature enough.In this study,CRISPR / Cas9 technique was used to explore the feasibility of spot integration of PRL,DGAT1 and CSN1S1.First,the screening gene Neo was added to the gene targeting vector pUC19-DS1-PRL-DS2,and the transfection conditions of buffalo kidney fibroblasts were explored and screened.The cleavage activity of three CRISPR / Cas9-1,CRISPR / Cas9-2 and CRISPR / Cas9-3 designed for the r DNA genomic loci of the buffalo was studied by T7E1 and clone sequencing.Followed by co-transfection of CRISPR / Cas9 and 3 gene targeting vectors,p UC19-DS1-PRL-2A-Neo-DS2,pUC19-DS1-DGAT1-2A-Zeocin-DS2,and pUC19-DS1-shRNA-2A-Neo-DS2,And detecting the gene integration and site-specific integration cases.The results showed that the gene was successfully added to the gene expression vector.The optimal screening concentration of G418 in buffalo fibroblasts was 400 ?g / ml.In the detection of CRISPR / Cas9 activity,it was found that the cutting efficiency was above 60.0%,up to 70.0%,and the gene locus appeared different degree of base insertion loss and mutation.In the three genes targeting vector gene integration test,found that genes are integrated.In order to further determine the integration site of the gene,we found that the PRL gene was integrated at the time of HRDS1 homologous arm sequencing,and the DGAT1 gene was found to be integrated at the time of cross-HRDS2 homologous arm sequencing,whereas the shRNA against CSN1S1 gene was not detected Fixed point integration.In summary,CRISPR / Cas9 can effectively mediate the integration of genes in the rDNA locus of buffalo.