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Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells

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Book cover Eukaryotic Transcriptional and Post-Transcriptional Gene Expression Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1507))

Abstract

CRISPR-Cas9 effector systems have wide applications for the stem cell and regenerative medicine field. The ability to dissect the functional gene regulatory networks in pluripotency and potentially in differentiation intermediates of all three germ layers makes this a valuable tool for the stem cell community. Catalytically inactive Cas9 fused to transcriptional/chromatin effector domains allows for silencing or activation of a genomic region of interest. Here, we describe the application of an inducible, RNA-guided, nuclease-deficient (d) Cas9-KRAB system (adapted from Streptococcus pyogenes) to silence target gene expression in human embryonic stem cells, via KRAB repression at the promoter region. This chapter outlines a detailed protocol for generation of a stable human embryonic stem cell line containing both Sp-dCas9-KRAB and sgRNA, followed by inducible expression of Sp-dCas9-KRAB to analyze functional effects of dCas9-KRAB at target loci in human embryonic stem cells.

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Acknowledgments

We thank Scot Wolfe and the members of the Maehr Lab for the helpful discussion and technical advice. R. M. is supported by The Leona M. and Harry B. Helmsley Charitable Trust (2012PG-T1D026 and 2015PG-T1D035), the Charles H. Hood Foundation Child Health Research Award, the Glass Family Charitable Foundation, and NIH grants 1R56AI114525 and 1R21AI119885.

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Authors and Affiliations

  1. Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, 01605, USA

    Krishna Mohan Parsi, Erica Hennessy, Nicola Kearns & René Maehr

Authors
  1. Krishna Mohan Parsi
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  2. Erica Hennessy
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  3. Nicola Kearns
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  4. René Maehr
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Corresponding author

Correspondence to René Maehr .

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Editors and Affiliations

  1. Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA

    Narendra Wajapeyee

  2. Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA

    Romi Gupta

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Parsi, K.M., Hennessy, E., Kearns, N., Maehr, R. (2017). Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells. In: Wajapeyee, N., Gupta, R. (eds) Eukaryotic Transcriptional and Post-Transcriptional Gene Expression Regulation. Methods in Molecular Biology, vol 1507. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6518-2_16

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  • DOI: https://doi.org/10.1007/978-1-4939-6518-2_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6516-8

  • Online ISBN: 978-1-4939-6518-2

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