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Abstract Abstract 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.
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Received: 10 January 2018
Published: 20 July 2018
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