The Study On Efficiency Of CRISPR/CAS9 And TALENs Mediated Gene Targeting At Dariy Goat β-Casein Locus

CRISPR/Cas9 is a newly developed gene editing technology, which can introduce double strand break (DSB) by Cas9 that guided by sgRNA to specific gene locus. Compared to ZFNs and TALENs, CRISPR/Cas9 not only has a great advantage by avoiding the recognition DNA sequence by structural identification, but also more efficient and easier to operate and obtain homozygous mutants. It can simultaneously introduce multiple mutations at different sites.In this study, a set of TALENs and a CRJSPR/Cas9 plasmid were designed for editing the dairy goat fi-casein (CSN2) gene at exon2, which is the first coding exon. Two targeting donor plasmids aimed to the TALENs and CRISPR/Cas9 site, named pFlrkt and pGlrkt, were constructed on the basis of GFP-PGK-Neor plasmid. In the 1024bp 5’homologous arm was located at the upstream of CAGG promoter, and 1028bp STiomologous arm located at the downstream of Neor gene. The exogenous gene was hFat-1 and EGFP, respectively. The structure of the recombinant plasmids was verified by restriction fragment analysis and DNA sequencing.Next, to test the efficiency TALENs and CRISPR/Cas9 mediated gene knock-in, the EGFP and hfat-1 expression plasmids pFlrkt and pGlrkt were co-transfected by electroporation to dairy goat fetal fibroblasts with TALENs and CRISPR/Cas9 plasmids. After G418 selection, cells colonies by cell shoved, the remaining cells picked monoclonal cell by using flow sorting method and screened by PCR, acrossed the homologous arms. Total 18 hfat-1 and 11 EGFP integrated cell lines were obtained from 68 pFlrkt transfected cell lines and 34 pGlrkt transfected cell lines by TALENs, the efficiency of gene knock-in of TALENs was 26.47% and 32.35%, respectively; 5 hfat-1 integrated cell lines were obtained from 23 pFlrkt transfected cell lines by using flow sorting method, the efficiency of gene knock-in of TALENs was 21.74%; 26 hfat-1 and 19 EGFP integrated cell lines were obtained from 35 pFlrkt transfected cell lines and 27 pGlrkt transfected cell lines by CRISPR/Cas9. The efficiency of gene knock-in of CRISPR/Cas9 was 74.29% and 70.37%, respectively; 10 hfat-1 integrated cell lines were obtained from 17 pFlrkt transfected cell lines by using flow sorting method, the efficiency of gene knock-in of CRISPR/Cas9 was 58.82%. The knock-in efficiency of CRISPR/Cas9 is higher than TALENs.In total,59 hfat-1 knock-in cell lines were obtained These cells will used as donor cells for subsequent somatic cell nuclear transfer and embryo transfer. Obtaining such β-casein gene knock-out dairy goat breeds can improve the quality of goat milk and reduce the difficulty of milk processing.