Yan-Yan Wei1, Qun-Mei Zhan1, Xiang-Xing Zhu2, Ai-Fen Yan1,3, Juan Feng3, Lian Liu3, Jian-Hao Li4, Dong-Sheng Tang5,6. 1. Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan, 528225, China. 2. Guangdong Provincial Engineering and Technology Research Center for Gene Editing, School of Medical Engineering, Foshan University, Foshan, 528225, China. zhu_xiangxing@126.com. 3. Guangdong Provincial Engineering and Technology Research Center for Gene Editing, School of Medical Engineering, Foshan University, Foshan, 528225, China. 4. State Key Laboratory of Livestock and Poultry Breeding, Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China. jianhao63@sina.com. 5. Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan, 528225, China. tangdsh@163.com. 6. Guangdong Provincial Engineering and Technology Research Center for Gene Editing, School of Medical Engineering, Foshan University, Foshan, 528225, China. tangdsh@163.com.
Abstract
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.
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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