Efficient CRISPR/Cas9-mediated gene editing in Guangdong small-ear spotted pig cells using an optimized electrotransfection method

文献类型： 外文期刊

第一作者： Wei, Yan-yan

作者： Wei, Yan-yan;Zhan, Qun-mei;Yan, Ai-fen;Tang, Dong-sheng;Zhu, Xiang-xing;Yan, Ai-fen;Feng, Juan;Liu, Lian;Tang, Dong-sheng;Li, Jian-hao

作者机构：

关键词： CRISPR; Cas9-mediated gene editing; Myostatin (MSTN); Insulin-like growth factor 2 (IGF2); Guangdong small-ear spotted (GDSS) pig; Electrotransfection

期刊名称：BIOTECHNOLOGY LETTERS （ 影响因子：2.461； 五年影响因子：2.457 ）

ISSN： 0141-5492

年卷期： 2020 年 42 卷 11 期

页码：

收录情况： SCI

摘要： Objectives Guangdong Small-ear Spotted (GDSS) pigs are a pig breed native to China that possesses unfortunate disadvantages, such as slow growth rate, low lean-meat percentage, and reduced feed utilization. In contrast to traditional genetic breeding methods with long cycle time and high cost, CRISPR/Cas9-mediated gene editing for the modification of the pig genome can quickly improve production traits, and therefore this technique exhibits important potential in the genetic improvement and resource development of GDSS pigs. In the present study, we aimed to establish an efficient CRISPR/Cas9-mediated gene-editing system for GDSS pig cells by optimizing the electrotransfection parameters, and to realize efficient CRISPR/Cas9-mediated gene editing of GDSS pig cells. Results After optimization of electrotransfection parameters for the transfection of GDSS pig cells, we demonstrated that a voltage of 150 V and a single pulse with a pulse duration of 20 ms were the optimal electrotransfection parameters for gene editing in these cells. In addition, our study generated GDSS pig single-cell colonies with biallelic mutations in the myostatin (MSTN) gene and insulin-like growth factor 2 (IGF2) intron-3 locus, which play an important role in pig muscle growth and muscle development. The single-cell colonies showed no foreign gene integration or off-target effects, and maintained normal cell morphology and viability. These gene-edited, single-cell colonies can in the future be used as donor cells to generate MSTN- and IGF2-edited GDSS pigs using somatic cell nuclear transfer (SCNT). Conclusions This study establishes the foundation for genetic improvement and resource development of GDSS pigs using CRISPR/Cas9-mediated gene editing combined with SCNT.

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