Pooled CRISPR screens with imaging on microraft arrays reveals stress granule-regulatory factors.

Autor: Wheeler EC; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine and UCSD Stem Cell Program, University of California San Diego, La Jolla, CA, USA., Vu AQ; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine and UCSD Stem Cell Program, University of California San Diego, La Jolla, CA, USA., Einstein JM; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine and UCSD Stem Cell Program, University of California San Diego, La Jolla, CA, USA., DiSalvo M; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill and Raleigh, NC, USA., Ahmed N; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine and UCSD Stem Cell Program, University of California San Diego, La Jolla, CA, USA., Van Nostrand EL; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine and UCSD Stem Cell Program, University of California San Diego, La Jolla, CA, USA., Shishkin AA; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine and UCSD Stem Cell Program, University of California San Diego, La Jolla, CA, USA.; Eclipse BioInnovations, San Diego, CA, USA., Jin W; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine and UCSD Stem Cell Program, University of California San Diego, La Jolla, CA, USA., Allbritton NL; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill and Raleigh, NC, USA.; Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.; Department of Bioengineering, University of Washington, Seattle, WA, USA., Yeo GW; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.; Institute for Genomic Medicine and UCSD Stem Cell Program, University of California San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.
Jazyk: angličtina
Zdroj: Nature methods [Nat Methods] 2020 Jun; Vol. 17 (6), pp. 636-642. Date of Electronic Publication: 2020 May 11.
DOI: 10.1038/s41592-020-0826-8
Abstrakt: Genetic screens using pooled CRISPR-based approaches are scalable and inexpensive, but restricted to standard readouts, including survival, proliferation and sortable markers. However, many biologically relevant cell states involve cellular and subcellular changes that are only accessible by microscopic visualization, and are currently impossible to screen with pooled methods. Here we combine pooled CRISPR-Cas9 screening with microraft array technology and high-content imaging to screen image-based phenotypes (CRaft-ID; CRISPR-based microRaft followed by guide RNA identification). By isolating microrafts that contain genetic clones harboring individual guide RNAs (gRNA), we identify RNA-binding proteins (RBPs) that influence the formation of stress granules, the punctate protein-RNA assemblies that form during stress. To automate hit identification, we developed a machine-learning model trained on nuclear morphology to remove unhealthy cells or imaging artifacts. In doing so, we identified and validated previously uncharacterized RBPs that modulate stress granule abundance, highlighting the applicability of our approach to facilitate image-based pooled CRISPR screens.
Databáze: MEDLINE
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