Pooled in vitro and in vivo CRISPR-Cas9 screening identifies tumor suppressors in human colon organoids
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 transfor...
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| Hauptverfasser: | , , , , , , , , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
7 May 2020
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| In: |
Cell stem cell
Year: 2020, Jahrgang: 26, Heft: 5, Pages: 782-792 |
| ISSN: | 1875-9777 |
| DOI: | 10.1016/j.stem.2020.04.003 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1016/j.stem.2020.04.003 Verlag, lizenzpflichtig, Volltext: http://www.sciencedirect.com/science/article/pii/S1934590920301429 
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| Verfasserangaben: | Birgitta E. Michels, Mohammed H. Mosa, Barbara I. Streibl, Tianzuo Zhan, Constantin Menche, Khalil Abou-El-Ardat, Tahmineh Darvishi, Ewelina Członka, Sebastian Wagner, Jan Winter, Hind Medyouf, Michael Boutros, Henner F. Farin |
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| 245 | 1 | 0 | |a Pooled in vitro and in vivo CRISPR-Cas9 screening identifies tumor suppressors in human colon organoids |c Birgitta E. Michels, Mohammed H. Mosa, Barbara I. Streibl, Tianzuo Zhan, Constantin Menche, Khalil Abou-El-Ardat, Tahmineh Darvishi, Ewelina Członka, Sebastian Wagner, Jan Winter, Hind Medyouf, Michael Boutros, Henner F. Farin |
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| 520 | |a 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 β (TGF-β) 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 pan-cancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC−/−;KRASG12D 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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| 650 | 4 | |a pooled-barcoded CRISPR-Cas9 screening | |
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| 650 | 4 | |a tumor suppressor genes | |
| 650 | 4 | |a unique molecular identifiers | |
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