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A Whole Genome CRISPR/Cas9 Screening Approach for Identifying Genes Encoding DNA End-Processing Proteins

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DNA Damage Responses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2444))

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Abstract

DNA double-strand breaks (DSBs) are mainly repaired by homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of HR or NHEJ is dictated in part by whether the broken DNA ends are resected to generate extended single-stranded DNA (ssDNA) overhangs, which are quickly bound by the trimeric ssDNA binding complex RPA, the first step of HR. Here we describe a series of protocols for generating Abelson murine leukemia virus-transformed pre-B cells (abl pre-B cells) with stably integrated inducible Cas9 that can be used to identify and study novel pathways regulating DNA end processing. These approaches involve gene inactivation by CRISPR/Cas9, whole genome guide RNA (gRNA) library-mediated screen, and flow cytometry-based detection of chromatin-bound RPA after DNA damage.

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Acknowledgments

B.P.S. is supported by National Institutes of Health grants R01 AI047829 and R01 AI074953.

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Authors and Affiliations

  1. Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

    Bo-Ruei Chen & Barry P. Sleckman

  2. O’Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA

    Bo-Ruei Chen & Barry P. Sleckman

Authors
  1. Bo-Ruei Chen
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  2. Barry P. Sleckman
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Corresponding author

Correspondence to Bo-Ruei Chen .

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Editors and Affiliations

  1. Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA

    Nima Mosammaparast

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Chen, BR., Sleckman, B.P. (2022). A Whole Genome CRISPR/Cas9 Screening Approach for Identifying Genes Encoding DNA End-Processing Proteins. In: Mosammaparast, N. (eds) DNA Damage Responses. Methods in Molecular Biology, vol 2444. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2063-2_2

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  • DOI: https://doi.org/10.1007/978-1-0716-2063-2_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2062-5

  • Online ISBN: 978-1-0716-2063-2

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