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Precise editing of myostatin signal peptide by CRISPR/Cas9 increases the muscle mass of Liang Guang Small Spotted pigs

  • Ruiqiang Li
  • Wu Zeng
  • Miao Ma
  • Zixuan Wei
  • Hongbo Liu
  • Xiaofeng Liu
  • Min Wang
  • Xuan Shi
  • Jianhua Zeng
  • Linfang Yang
  • Delin Mo
  • Xiaohong Liu
  • Yaosheng Chen
  • Zuyong HeEmail author
Original Paper

Abstract

Myostatin (MSTN), a member of the transforming growth factor-β superfamily, is a negative regulator of muscle growth and development. Disruption of the MSTN gene in various mammalian species markedly promotes muscle growth. Previous studies have mainly focused on the disruption of the MSTN peptide coding region in pigs but not on the modification of the signal peptide region. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system was used to successfully introduce two mutations (PVD20H and GP19del) in the MSTN signal peptide region of the indigenous Chinese pig breed, Liang Guang Small Spotted pig. Both mutations in signal peptide increased the muscle mass without inhibiting the production of mature MSTN peptide in the cells. Histological analysis revealed that the enhanced muscle mass in MSTN+/PVD20H pig was mainly due to an increase in the number of muscle fibers. The expression of MSTN in the longissimus dorsi muscle of MSTN+/PVD20H and MSTNKO/PVD20H pigs was significantly downregulated, whereas that of myogenic regulatory factors, including MyoD, Myogenin, and Myf-5, was significantly upregulated when compared to those in the longissimus dorsi muscle of wild-type pigs. Meanwhile, the mutations also activated the PI3K/Akt pathway. The results of this study indicated that precise editing of the MSTN signal peptide can enhance porcine muscle development without markedly affecting the expression of mature MSTN peptide, which could exert other beneficial biological functions in the edited pigs.

Keywords

Myostatin Signal peptide CRISPR/Cas9 Muscle mass Liang Guang Small Spotted pig 

Notes

Acknowledgements

We wish to thank past and current members of Prof. YC’s group for scientific discussions. We are particular grateful to the staffs in the Guangdong YIHAO Food Co., Ltd. for their help in animal sample collection.

Funding

This work was jointly supported by the National Transgenic Major Program of China (2016ZX08006003-006), National Key R&D Programmes of China (2018YFD0501200), The Key R&D Programmes of Guangdong Province (2018B020203003) and Guangdong Basic and Applied Basic Research Foundation (2019A1515011134).

Compliance with ethical standards

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC), Sun Yat-sen University (Approval Number: IACUC DD-17-0403).

Supplementary material

11248_2020_188_MOESM1_ESM.docx (16 kb)
Supplementary file1 (DOCX 15 kb)

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Copyright information

© Springer Nature Switzerland AG 2020

Authors and Affiliations

  • Ruiqiang Li
    • 1
  • Wu Zeng
    • 1
  • Miao Ma
    • 1
  • Zixuan Wei
    • 1
  • Hongbo Liu
    • 1
  • Xiaofeng Liu
    • 1
  • Min Wang
    • 1
  • Xuan Shi
    • 1
  • Jianhua Zeng
    • 2
  • Linfang Yang
    • 2
  • Delin Mo
    • 1
  • Xiaohong Liu
    • 1
  • Yaosheng Chen
    • 1
  • Zuyong He
    • 1
    Email author
  1. 1.State Key Laboratory of Biocontrol, School of Life SciencesSun Yat-sen UniversityGuangzhouPeople’s Republic of China
  2. 2.Guangdong YIHAO Food Co., Ltd.GuangzhouPeople’s Republic of China

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