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A Method to Map Gene Essentiality of Human Pluripotent Stem Cells by Genome-Scale CRISPR Screens with Inducible Cas9

  1. Author:
    Mair, Barbara
    Aregger,Michael
    Tong, Amy H Y
    Chan, Katherine S K
    Moffat, Jason

  2. Author Address

    Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada., Boehringer Ingelheim, Vienna, Austria., Center for Cancer Research, National Cancer Institute, Frederick, MD, USA., Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada. j.moffat@utoronto.ca., Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. j.moffat@utoronto.ca., Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada. j.moffat@utoronto.ca.,
    1. Year: 2022
  2. Journal: Methods in molecular biology (Clifton, N.J.)
    1. 2377
    2. Pages: 1-27
  4. Type of Article: Article
  1. Abstract:

    Human pluripotent stem cells (hPSCs) have the capacity for self-renewal and differentiation into most cell types and, in contrast to widely used cell lines, are karyotypically normal and non-transformed. Hence, hPSCs are considered the gold-standard system for modelling diseases, especially in the field of regenerative medicine. Despite widespread research use of hPSCs and induced pluripotent stem cells (iPSCs), the systematic understanding of pluripotency and lineage differentiation mechanisms are still incomplete. Before tackling the complexities of lineage differentiation with genetic screens, it is critical to catalogue the general genetic requirements for cell fitness and proliferation in the pluripotent state and assess their plasticity under commonly used culture conditions.We describe a method to map essential genetic determinants of hPSC fitness and pluripotency, herein defined as cell reproduction, by genome-scale loss-of-function CRISPR screens in an inducible S. pyogenes Cas9 H1 hPSC line. To address questions of context-dependent gene essentiality, we include protocols for screening hPSCs cultured on feeder cells and laminin, two commonly used growth substrates. This method establishes parameters for genome-wide screens in hPSCs, making human stem cells amenable for functional genomics approaches to facilitate investigation of hPSC biology. © 2022. Springer Science+Business Media, LLC, part of Springer Nature.

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  2. DOI: 10.1007/978-1-0716-1720-5_1
  3. PMID: 34709608

Library Notes

  1. Fiscal Year: FY2021-2022
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