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Genome-Wide CRISPR Screening to Identify Mammalian Factors that Regulate Intron Retention

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Alternative Splicing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2537))

Abstract

Intron retention (IR) regulates gene expression to control fundamental biological processes like metabolism, differentiation, and cell cycle. Despite a wide variety of genes controlled by IR, few techniques are available to identify regulators of IR in an unbiased manner. Here, we describe a CRISPR knockout screening method that can be applied to uncover regulators of IR. This method uses GFP reporter constructs containing a retained intron from a gene of interest such that GFP signal is regulated by IR in the same fashion as the endogenous gene. The GFP levels are then used as a readout for genome-wide CRISPR screening. We have successfully used this approach to identify novel regulator of IR of the MAT2A transcript and propose that similar screens will be broadly applicable for the identification of novel factors that control IR of specific transcripts.

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Acknowledgments

We would like to thank Dr. John Schoggins and Dr. Joshua Mendell and their lab members for their invaluable guidance in setting up CRISPR screens. This work was supported by the National Institutes of Health R01 GM127311, U01 CA242115, and T32 GM007062.

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Authors and Affiliations

  1. Department of Microbiology, UT Southwestern Medical Center, Dallas, TX, USA

    Anna M. Scarborough, Ashwin Govindan & Nicholas K. Conrad

Authors
  1. Anna M. Scarborough
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  2. Ashwin Govindan
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  3. Nicholas K. Conrad
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Corresponding author

Correspondence to Nicholas K. Conrad .

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Editors and Affiliations

  1. Biozentrum, University of Basel, Basel, Switzerland

    Peter Scheiffele

  2. Biozentrum, University of Basel, Basel, Switzerland

    Oriane Mauger

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Scarborough, A.M., Govindan, A., Conrad, N.K. (2022). Genome-Wide CRISPR Screening to Identify Mammalian Factors that Regulate Intron Retention. In: Scheiffele, P., Mauger, O. (eds) Alternative Splicing. Methods in Molecular Biology, vol 2537. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2521-7_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2521-7_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2520-0

  • Online ISBN: 978-1-0716-2521-7

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