Journal
CELL STEM CELL
Volume 26, Issue 5, Pages 782-+Publisher
CELL PRESS
DOI: 10.1016/j.stem.2020.04.003
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Funding
- German Cancer Aid [70112869]
- LOEWE Center Frankfurt Cancer Institute (FCI) - Hessen State Ministry of Higher Education, Research and the Arts [III L 5-519/03/03.001-(0015)]
- German Jose Carreras Leukemia Foundation [DJCLS 04FN/2018]
- Clinician Scientist program Interfaces and Interventions in Complex Chronic Conditions'' by the German Research Foundation (DFG)
- ERC-starting grant [639795]
- German Cancer Consortium (DKTK) Joint Funding (CHOICE consortium)
- DFG [SPP 2084]
- European Research Council (ERC) [639795] Funding Source: European Research Council (ERC)
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Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneity between patients. To facilitate high-throughput genetic testing and functional identification of tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in human colon organoids. Using transforming growth factor beta (TGF-beta) resistance as a paradigm to establish sensitivity and scalability in vitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screening in 3D organoids. We then screened a pancancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC(-/-);KRAS(G12D) mutations, which were xenografted to study clonal advantages in context of a complex tumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediators of CRC growth. gRNAs were validated in a secondary screen using unique molecular identifiers (UMIs) to adjust for clonal drift and to distinguish clone size and abundance. Together, these findings highlight a powerful organoid-based platform for pooled CRISPR-Cas9 screening for patient-specific functional genomics.
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