Editing MSTN gene of Guangdong Xiaoerhua pig by using CRISPR/Cas9

MSTN（ Myostatin） gene functions as a negative regulator of muscle growth. Therefore，disrupting the expression of MSTN has been proved as an efficient strategy to improve the muscle mass in livestock and poultry. In this study，we edited the exons of MSTN gene to disrupt its expression in order to increase muscle production of Guangdong Xiaoerhua pig by using the cutting-edge genome editing technology—CRISPR/Cas9. We designed six guide RNAs targeting on loci across three exons of MSTN gene. Through T7E1 assay and TA cloning analysis，we found that three guide RNAs were capable of targeted cutting on MSTN gene efficiently， with the targeting efficiency ranging from 17.0% to 28.3%. The editing efficiency of gRNA1-1 and gRNA3-3 in PEF cells of Guangdong Xiaoerhua pig，were both up to 50%. Particularly，editing efficiency in the third exon was as high as 61.5%. This study provides basis to generate MSTN-edited Xiaoerhua pigs by somatic cell nuclease transfer（SCNT） in future.