肌肉生长抑制素（myostatin, MSTN）是肌肉生长负调控因子，MSTN基因突变可造成MSTN生物学功能缺陷，从而引起动物的双肌性状。本研究旨在筛选靶向绵羊（Ovis aries）MSTN基因的高效单导向RNA（single-guide RNA, sgRNA），并构建可标记表达载体，以提高CRISPR/Cas9（clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9）介导的绵羊MSTN基因突变效率。利用Gibson Assembly法将增强绿色荧光蛋白（enhanced green fluorescent protein, EGFP）序列与串联表达原件2A序列插入pX330质粒载体中，构建pX330-EGFP质粒载体，并将设计好的12个sgRNA寡核苷酸链以Golden Gate法分别连入pX330-EGFP质粒载体，以构建12个不同的pX330-EGFP-sgRNA质粒表达载体；将构建好的pX330-EGFP-sgRNA表达载体以电转染的方法分别转入12组绵羊成纤维细胞，48 h后提取各组部分细胞基因组，利用SURVEROR分析初选获得3个具有靶向效果的细胞群（T2, T9, Q2），其对应转染质粒为pX330-EGFP-sgRNA；之后分别提取3组细胞中的阳性转染细胞基因组，扩增MSTN基因并进行测序分析，得出sgRNA-T2、sgRNA-T9、sgRNA-Q2的打靶效率分别为40%、40%、60%。本研究通过构建表达绿色荧光蛋白（green fluorescent protein, GFP）的pX330-EGFP-sgRNA表达载体，成功筛选到高效靶向绵羊MSTN基因的sgRNA，并获得准确的编辑效率，为后续其他基因sgRNA的筛选提供了借鉴，并为MSTN基因编辑羊的生产提供科学依据。
Myostatin （MSTN） is a negative regulator to muscle cells growth and differentiation. MSTN gene＇s mutation will lost its function, which will make animals have significantly more muscle mass. The purposes of this study are to find the best sgRNA which could edit sheep＇s （Ovis aries） MSTN gene efficiently and build EGFP and sgRNA co-expression vectors, with which CRISPR/Cas9 system could improve sheep MSTN gene＇s editing efficiency. First using Gibson Assembly method to incorporate the 2A＋ enhanced green fluorescent protein （EGFP） into pX330, the study got the pX330-EGFP vector. Then 12 sgRNAs were designed and using Golden Gate method separately, inserted these single-guide RNA （sgRNA） oligonucleotides into pX330-EGFP plasmid, and 12 pX330-EGFP-sgRNA expression plasmids were got. The 12 pX330-EGFP-sgRNA vectors were transferred into sheep fibroblasts by electroporation. After 48 h, using SURVEROR analysis, it was be found that 3 groups （T2, T9, Q2） of cell＇s DNA were edited, which were groups of sgRNA. Then 100 green cells of each group were collected and extracted the DNA, after amplification of the MSTN gene by PCR, the productions were send for sequencing analysis. The results showed that the targeting efficiency of sgRNA-T2, sgRNA-T9 and sgRNA-Q2 were 40%, 40% and 60% respectively. In this study, we build EGFP and sgRNA co-expression vetors and selected the best sgRNA to sheep MSTN gene which was 60%. This protocol will be helpful to find more sgRNAs to different genes. These results provide a scientific basic for the production of MSTN gene editing sheep.
Journal of Agricultural Biotechnology