RNA Binding Proteins As Regulators of Oxidative Stress Identified by a Targeted CRISPR-Cas9 Single Guide RNA Library.

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  • Author(s): Turner DJ;Turner DJ; Turner M; Turner M
  • Source:
    The CRISPR journal [CRISPR J] 2021 Jun; Vol. 4 (3), pp. 427-437. Date of Electronic Publication: 2021 Jun 04.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Mary Ann Liebert, Inc Country of Publication: United States NLM ID: 101738191 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2573-1602 (Electronic) Linking ISSN: 25731599 NLM ISO Abbreviation: CRISPR J Subsets: MEDLINE
    • Publication Information:
      Original Publication: [New Rochelle, NY] : Mary Ann Liebert, Inc., [2018]-
    • Subject Terms:
      CRISPR-Cas Systems*; Oxidative Stress/*physiology ; RNA, Guide, CRISPR-Cas Systems/*genetics ; RNA-Binding Proteins/*metabolism; Cell Line, Tumor ; DNA Helicases/genetics ; Eukaryotic Initiation Factor-4E ; Gene Knockout Techniques ; Genomic Library ; Humans ; Poly-ADP-Ribose Binding Proteins/genetics ; RNA Helicases/genetics ; RNA Recognition Motif Proteins/genetics ; RNA-Binding Proteins/genetics
    • Abstract:
      The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing system has been broadly adopted for high-throughput genetic screens. However, the application of genome-wide single guide RNA (sgRNA) libraries can be challenging. We generated a custom sgRNA library, an order of magnitude smaller than genome-wide alternatives, to facilitate the genetic screening of RNA binding proteins (RBPs). We demonstrated the utility of our reagent in a genetic screen for RBPs that conveyed cellular resistance or sensitivity to oxidative stress induced by paraquat. This identified that CSDE1 and STRAP, proteins that interact with each other, convey sensitivity to oxidative stress and that Pumilio homologues (PUM1 and PUM2) convey resistance. Targeting eIF4-E1 and -A1 protected cells from high-dose paraquat, whereas eIF4E2 targeted cells did less well. We also found that G3BP1 promoted sensitivity to a low dose of paraquat but protected cells at a higher dose. Our study highlights the use of genetic screens to identify roles of RBPs and identifies novel genes regulating sensitivity to oxidative stress.
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    • Grant Information:
      BBS/E/B/000C0407 United Kingdom BB_ Biotechnology and Biological Sciences Research Council; 200823/Z/16/Z United Kingdom WT_ Wellcome Trust
    • Accession Number:
      0 (EIF4E2 protein, human)
      0 (Eukaryotic Initiation Factor-4E)
      0 (PUM1 protein, human)
      0 (PUM2 protein, human)
      0 (Poly-ADP-Ribose Binding Proteins)
      0 (RNA Recognition Motif Proteins)
      0 (RNA, Guide, CRISPR-Cas Systems)
      0 (RNA-Binding Proteins)
      EC 3.6.4.- (DNA Helicases)
      EC 3.6.4.12 (G3BP1 protein, human)
      EC 3.6.4.13 (RNA Helicases)
    • Publication Date:
      Date Created: 20210607 Date Completed: 20211221 Latest Revision: 20240306
    • Publication Date:
      20240306
    • Accession Number:
      PMC8236562
    • Accession Number:
      10.1089/crispr.2020.0116
    • Accession Number:
      34096786