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CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium

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In Vitro Mutagenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1498))

Abstract

The recent advent of engineered nucleases including the CRISPR/Cas9 system has greatly facilitated genome manipulation in human pluripotent stem cells (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clinical translation, it is necessary to use a defined, xeno-free cell culture system. This chapter describes protocols for CRISPR/Cas9 genome editing in an inducible Cas9 hPSC-based system, using cells cultured in chemically defined, xeno-free E8 Medium on a recombinant human vitronectin substrate. We detail procedures for the design and transfection of CRISPR guide RNAs, colony selection, and the expansion and validation of clonal mutant lines, all within this fully defined culture condition. These methods may be applied to a wide range of genome-engineering applications in hPSCs, including those that utilize different types of site-specific nucleases such as zinc finger nucleases (ZFNs) and TALENs, and form a closer step towards clinical utility of these cells.

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Acknowledgments

Our work was funded in part by NIH (R01DK096239), NYSTEM (C029156), and March of Dimes Foundation (Basil O’Connor Starter Scholar Research Award Grant 5-FY12-82).

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Authors and Affiliations

  1. Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA

    Chew-Li Soh & Danwei Huangfu

Authors
  1. Chew-Li Soh
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  2. Danwei Huangfu
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Corresponding author

Correspondence to Danwei Huangfu .

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Editors and Affiliations

  1. Coskata, Inc., Warrenville, Illinois, USA

    Andrew Reeves

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Soh, CL., Huangfu, D. (2017). CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium. In: Reeves, A. (eds) In Vitro Mutagenesis. Methods in Molecular Biology, vol 1498. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6472-7_5

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  • DOI: https://doi.org/10.1007/978-1-4939-6472-7_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6470-3

  • Online ISBN: 978-1-4939-6472-7

  • eBook Packages: Springer Protocols

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