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CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells

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Cancer Gene Networks

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1513))

Abstract

Fluorescent reporter and epitope-tagged human pluripotent stem cells (hPSCs) greatly facilitate studies on the pluripotency and differentiation characteristics of these cells. Unfortunately traditional procedures to generate such lines are hampered by a low targeting efficiency that necessitates a lengthy process of selection followed by the removal of the selection cassette. Here we describe a procedure to generate fluorescent reporter and epitope tagged hPSCs in an efficient one-step process using the CRISPR/Cas technology. Although the method described uses our recently developed iCRISPR platform, the protocols can be adapted for general use with CRISPR/Cas or other engineered nucleases. The transfection procedures described could also be used for additional applications, such as overexpression or lineage tracing studies.

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Acknowledgements

Nipun Verma and Zengrong Zhu contributed equally to this work. Our work related to this publication was funded, in part, by NIH (R01DK096239) and NYSTEM (C029156). Z.Z. was supported by the New York State Stem Cell Science (NYSTEM) fellowship from the Center for Stem Cell Biology (CSCB) of the Sloan Kettering Institute. N.V. was supported by the Howard Hughes Medical Institute (HHMI) Medical Research and the Tri-Institutional Weill Cornell/ Rockefeller/ Sloan Kettering MD-PhD program.

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    Authors and Affiliations

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

      Nipun Verma, Zengrong Zhu & Danwei Huangfu

    2. Weill Graduate School of Medical Sciences at Cornell University/The Rockefeller University/Sloan Kettering Institute Tri-Institutional M.D.-Ph.D. Program, 1300 York Avenue, New York, NY, 10065, USA

      Nipun Verma

    Authors
    1. Nipun Verma
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    2. Zengrong Zhu
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    3. Danwei Huangfu
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    Corresponding author

    Correspondence to Danwei Huangfu .

    Editor information

    Editors and Affiliations

    1. Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA

      Usha Kasid

    2. Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA

      Robert Clarke

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    Verma, N., Zhu, Z., Huangfu, D. (2017). CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells. In: Kasid, U., Clarke, R. (eds) Cancer Gene Networks. Methods in Molecular Biology, vol 1513. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6539-7_9

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    • DOI: https://doi.org/10.1007/978-1-4939-6539-7_9

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    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-6537-3

    • Online ISBN: 978-1-4939-6539-7

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