The Study On Efficiency Of CRISPR/CAS9 And ZFNs Mediated Cattle MSTN Gene Targeting

CRISPR/Cas9 is a newly developed gene editing technology, which introduce double strand break (DSB) by Cas9 that guided by sgRNA to specific gene locus. CRISPR/Cas9 not only can introduce the fine change or silence at DNA and RNA level like ZFNs and TALENs, but also has a great advantage by avoiding the recognition DNA sequence by structural identification.In this study, a CRISPR/Cas9A plasmid was designed for editing the bovine myostatin(MSTTN)gene at a sequence of exon2, a set of ZFNs and a CRISPR/Cas9B plasmid were designed for editing the bovine MSTN gene at exon 2. The electroporation methods was used to transfect the bovine fetal fibroblast. Single cells were picked into 96-well plate by mouth pipetting after transfection. After PCR and sequencing,4 knockout cell colonies were obtained from 74 colonies by ZFNs, the knockdown efficiency was 5.12%; and 22 knockout cell colonies were detected from 77 colonies by CRISPR/Cas9B, which included 2 double knock out cell colonies that included the 3bp insertion and the 13bp deletion, respectively. The knockout and double knockout efficiency of CRISPR/Cas9B was 27.05% and 9.52%, respectively. The results indicated CRISPR/Cas9B was worked better than CRISPR/Cas9A, and CRISPR/Cas9 is more efficient than ZFNs to introduce bovine MSTN mutation.Next, to test the efficiency ZFNs and CRISPR/Cas9B mediated gene integration, the EGFP and hfatl expression plasmids pflrkt and pGlrkt were constructed, which both included the 885 bp 5’homologous arms and 823bp 3’homologous arms.The different sets of plasmids were co-transfected by electroporation transfection to bovine fetal fibroblasts. After G418 selection, cells colonies collected by cell shove, and screened by PCR across the homologous arms,13 EGFP integrated cell lines were obtained from 95 pGlrkt integrated cell lines by ZFNs; 64 hfat-1 and 74 EGFP integrated cell lines were obtained from 81 pflrkt transfected cell lines and 119 pGlrkt transfected cell lines by CRISPR/Cas9B. The gene knockin efficiency of CRISPR/Cas9 B was 79.01% and 57.84%, respectively. The gene knockin efficiency of CRISPR/Cas9B is about five times higher than ZFNs.In total,26 MSTN knock out bovine fetal fibroblast cell lines and 64 hfat-1 knock in cell lines were obtained, which is a valuable materials for bovine double muscle breeding. The experiment data showed the genome editing efficiency of CRISPR/Cas9 is better than that of ZFNs.