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Genome-scale CRISPR screening in human intestinal organoids identifies drivers of TGF-β resistance


Ringel, Till; Frey, Nina; Ringnalda, Femke; Janjuha, Sharan; Cherkaoui, Sarah; Butz, Stefan; Srivatsa, Sumana; Pirkl, Martin; Russo, Giancarlo; Villiger, Lukas; Rogler, Gerhard; Clevers, Hans; Beerenwinkel, Niko; Zamboni, Nicola; Baubec, Tuncay; Schwank, Gerald (2020). Genome-scale CRISPR screening in human intestinal organoids identifies drivers of TGF-β resistance. Cell Stem Cell, 26(3):431-440.e8.

Abstract

Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.

Abstract

Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Pharmacology and Toxicology
07 Faculty of Science > Institute of Pharmacology and Toxicology

04 Faculty of Medicine > University Hospital Zurich > Clinic for Gastroenterology and Hepatology
05 Vetsuisse Faculty > Veterinärwissenschaftliches Institut > Department of Molecular Mechanisms of Disease
07 Faculty of Science > Department of Molecular Mechanisms of Disease
Dewey Decimal Classification:610 Medicine & health
Scopus Subject Areas:Life Sciences > Molecular Medicine
Life Sciences > Genetics
Life Sciences > Cell Biology
Language:English
Date:5 March 2020
Deposited On:01 Sep 2020 15:51
Last Modified:23 Jul 2024 01:38
Publisher:Cell Press (Elsevier)
ISSN:1875-9777
OA Status:Hybrid
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1016/j.stem.2020.02.007
PubMed ID:32142663
  • Content: Published Version
  • Licence: Creative Commons: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)