应用CRISPR/Cas9编辑广东小耳花猪MSTN基因  

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

作　　者：王敏[1] 黄翔[1] 石翾 刘小凤[1] 曾检华 刘小红[1] 陈瑶生[1] 何祖勇[1] 

机构地区：[1]中山大学生命科学学院/有害生物控制与资源利用国家重点实验室,广东广州510006 [2]广东壹号食品股份有限公司,广东广州510620

出　　处：《广东农业科学》2017年第2期141-148,共8页Guangdong Agricultural Sciences

基　　金：国家转基因生物新品种培育重大专项(2016ZX08006003-006);广东省自然科学基金(2016A0 30313310);广东省扬帆计划项目(2014YT02H042)

摘　　要：MSTN基因是肌肉生长抑制基因,破坏MSTN基因的表达成为提高畜禽肌肉产量的有效途径。通过应用CRISRP/Cas9编辑技术对广东小耳花猪MSTN基因的外显子区域进行编辑,破坏MSTN基因的读码框来抑制其表达,从而提高广东小耳花猪的肌肉产量。共设计了6条靶向MSTN基因3个不同外显子的gRNA,经过T7E1酶切检测和TA克隆分析,发现其中3条gRNA能够发生有效切割,其中切割效率最高的达28.3%,最低为17.0%。gRNA1-1和gRNA3-3突变的广东小耳花猪PEF细胞系,突变效率均在50%以上,其中三号外显子上突变位点的突变率达到61.5%。试验结果为后续进行体细胞核移植,生产MSTN基因编辑的广东小耳花猪奠定了基础。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 gRNAI-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.

关 键 词：MSTN CIRSPR/Cas9 广东小耳花猪 基因编辑 

分 类 号：Q789[生物学—分子生物学] S813.3[农业科学—畜牧学]