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Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9

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Pluripotent Stem-Cell Derived Cardiomyocytes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2320))

Abstract

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.

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Acknowledgments

We would like to thank all the lab members in the Division of Regenerative Medicine, Jichi Medical University for technical supports and helpful discussions. The methods described here were developed with the funding supports from The Program for Technological Innovation of Regenerative Medicine, Research Center Network for Realization of Regenerative Medicine from Japan Agency for Medical Research and Development (AMED, JP18bm0704012), Fund for the Promotion of Joint International Research (Fostering Joint International Research (B), JP19KK0219) from Japan Society for the Promotion of Science, and the Grant for Basic Research of the Japanese Circulation Society (to HU). This study was also supported in part by the Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED (JP18am0301002 to Y.H. as co-PI).

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Author notes

  1. Tatsuya Anzai and Hiromasa Hara contributed equally to this work.

Authors and Affiliations

  1. Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan

    Tatsuya Anzai, Hiromasa Hara, Nawin Chanthra, Yutaka Hanazono & Hideki Uosaki

  2. Department of Pediatrics, Jichi Medical University, Tochigi, Japan

    Tatsuya Anzai

  3. Animal Resource Laboratory, Center for Development of Advanced Medical Technology, Jichi Medical University, Tochigi, Japan

    Hiromasa Hara

  4. Department of Cardiology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

    Taketaro Sadahiro & Masaki Ieda

  5. Translational Research Laboratory, Center for Development of Advanced Medical Technology, Jichi Medical University, Tochigi, Japan

    Yutaka Hanazono & Hideki Uosaki

Authors
  1. Tatsuya Anzai
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  2. Hiromasa Hara
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  3. Nawin Chanthra
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  4. Taketaro Sadahiro
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  5. Masaki Ieda
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  6. Yutaka Hanazono
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  7. Hideki Uosaki
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Corresponding author

Correspondence to Hideki Uosaki .

Editor information

Editors and Affiliations

  1. Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan

    Yoshinori Yoshida

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Anzai, T. et al. (2021). Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9. In: Yoshida, Y. (eds) Pluripotent Stem-Cell Derived Cardiomyocytes. Methods in Molecular Biology, vol 2320. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1484-6_22

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  • DOI: https://doi.org/10.1007/978-1-0716-1484-6_22

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1483-9

  • Online ISBN: 978-1-0716-1484-6

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