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The Integrated Stress Response pp 19–40Cite as

CRISPR-Based Screening for Stress Response Factors in Mammalian Cells

Part of the Methods in Molecular Biology book series (MIMB,volume 2428)

Abstract

In the presence of different physiological and environmental stresses, cells rapidly initiate stress responses to re-establish cellular homeostasis. Stress responses usually orchestrate both transcriptional and translational programs via distinct mechanisms. With the advance of transcriptomics and proteomics technologies, transcriptional and translational outputs to a particular stress condition have become easier to measure; however, these technologies lack the ability to reveal the upstream regulatory pathways. Unbiased genetic screens based on a transcriptional or translational reporter are powerful approaches to identify regulatory factors of a specific stress response. CRISPR/Cas-based technologies, together with next-generation sequencing, enable genome-scale pooled screens to systematically elucidate gene function in mammalian cells, with a significant reduction in the rate of off-target effects compared to the previously used RNAi technology. Here, we describe our fluorescence-activated cell sorting (FACS)-based CRISPR interference (CRISPRi) screening platform using a translational reporter to identify novel genetic factors of the mitochondrial stress response in mammalian cells. This protocol provides a general framework for scientists who wish to establish a reporter-based CRISPRi screening platform to address questions in their area of research.

Key words

  • CRISPRi
  • Genetic screens
  • Mitochondrial stress response
  • Transcriptional reporter
  • Translational reporter
  • FACS
  • Mammalian cells

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Acknowledgments

We thank many of the current (Lydia Lee, Sydney Sattler, Stephanie See, Nina Drager, Avi Samelson, Emmy Li, Kun Leng, Jaime Leong, Merissa Chen, Giovanni Aviles, Athony Abarientons, Brandan Rooney, Greg Mohl) and previous members (Diane Nathaniel, Connor Ludwig, Jason Hong, Poornima Ramkumar, John Chen, Ruilin Tian) of the Kampmann lab for optimizing and organizing many protocols included here. We thank Ruilin Tian and Avi Samelson for comments on the manuscript. This work was supported by the Larry L. Hillblom Foundation to X.G. and by the National Institutes of Health grants GM119139 to M.K.. M.K. is a Chan Zuckerberg Biohub Investigator.

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Authors and Affiliations

  1. Institute for Neurodegenerative Diseases, University of California, San Francisco, CA, USA

    Xiaoyan Guo & Martin Kampmann

  2. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Martin Kampmann

  3. Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA

    Martin Kampmann

Authors
  1. Xiaoyan Guo
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  2. Martin Kampmann
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Correspondence to Xiaoyan Guo or Martin Kampmann .

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Editors and Affiliations

  1. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

    Daniel Matějů

  2. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

    Dr. Jeffrey A. Chao

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Guo, X., Kampmann, M. (2022). CRISPR-Based Screening for Stress Response Factors in Mammalian Cells. In: Matějů, D., Chao, J.A. (eds) The Integrated Stress Response. Methods in Molecular Biology, vol 2428. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1975-9_2

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  • DOI: https://doi.org/10.1007/978-1-0716-1975-9_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1974-2

  • Online ISBN: 978-1-0716-1975-9

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