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.
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Acknowledgements
This work was jointly supported by the National Science and Technology Major Project (2009ZX08010-023B, 2018ZX08010-08B), Guangdong Provincial R&D Project in Key Areas (2018B020203003), Foshan Science and Technology Innovation Project (2017AG100111) and Open Project of Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding (2018A05), Guangdong Foundation and Applied Basic Research Fund (2019A1515110280), and Guangzhou Science and Technology Planning Project (201707020007).
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DST and XXZ conceived and designed the experiments. YYW, QMZ and XXZ conducted the experiments and analyzed the results. DST, XXZ, YYW and QMZ drafted the manuscript. JHL and all other authors participated in discussions of the results and revised the manuscript.
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Clarification on animal ethics
All of the animal procedures used in this study were approved by the Animal Care & Welfare Committee of Foshan University (approval no. 2019020). All of the surgical procedures were performed under anesthesia by a veterinarian, and all efforts were made to minimize animal suffering.
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Wei, Yy., Zhan, Qm., Zhu, Xx. et al. Efficient CRISPR/Cas9-mediated gene editing in Guangdong small-ear spotted pig cells using an optimized electrotransfection method. Biotechnol Lett 42, 2091–2109 (2020). https://doi.org/10.1007/s10529-020-02930-0
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DOI: https://doi.org/10.1007/s10529-020-02930-0