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CRISPR-Cas9-Mediated Gene Editing in Mouse Spermatogonial Stem Cells

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RNAi and Small Regulatory RNAs in Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1622))

Abstract

Precise genome editing is a powerful tool for analysis of gene function. However, in spermatogonial stem cells (SSCs), this still remains a big challenge mainly due to low efficiency and complexity of currently available gene editing techniques. The CRISPR-Cas9 system from bacteria has been applied to modifying genome in different species at a very high efficiency and specificity. Here we describe CRISPR-Cas9-mediated gene editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in SSCs. This protocol provides guidelines for derivation of SSCs, nucleofection of SSCs with the CRISPR-Cas9 system, transplantation of the gene-modified SSCs into the recipient testes, and production of mice using transplanted SSC-derived round spermatids.

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Acknowledgment

This study was supported by grants from the Ministry of Science and Technology of China (2014CB964803, 2015AA020307 and 2013CB967103), the National Natural Science Foundation of China (31530048 and 81672117), the Chinese Academy of Sciences (XDB19010204), and the Shanghai Municipal Commission for Science and Technology (16JC1420500).

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Authors and Affiliations

  1. State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, 200031, China

    Yinghua Wang, Yifu Ding & Jinsong Li

  2. School of Life Science and Technology, ShanghaiTech University, 100 Haike Road, Shanghai, 201210, China

    Jinsong Li

Authors
  1. Yinghua Wang
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  2. Yifu Ding
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  3. Jinsong Li
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Corresponding author

Correspondence to Jinsong Li .

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Editors and Affiliations

  1. Department of Biology, East Carolina University, Greenville, North Carolina, USA

    Baohong Zhang

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Wang, Y., Ding, Y., Li, J. (2017). CRISPR-Cas9-Mediated Gene Editing in Mouse Spermatogonial Stem Cells. In: Zhang, B. (eds) RNAi and Small Regulatory RNAs in Stem Cells. Methods in Molecular Biology, vol 1622. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7108-4_20

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  • DOI: https://doi.org/10.1007/978-1-4939-7108-4_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7106-0

  • Online ISBN: 978-1-4939-7108-4

  • eBook Packages: Springer Protocols

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