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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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Main Authors: Michels, Birgitta E. (Author) , Mosa, Mohammed H. (Author) , Streibl, Barbara I. (Author) , Zhan, Tianzuo (Author) , Menche, Constantin (Author) , Abou-El-Ardat, Khalil (Author) , Darvishi, Tahmineh (Author) , Członka, Ewelina (Author) , Wagner, Sebastian (Author) , Winter, Jan (Author) , Medyouf, Hind (Author) , Boutros, Michael (Author) , Farin, Henner F. (Author)
Format: Article (Journal)
Language:English
Published: 7 May 2020
In: Cell stem cell
Year: 2020, Volume: 26, Issue: 5, Pages: 782-792
ISSN:1875-9777
DOI:10.1016/j.stem.2020.04.003
Online Access: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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Author Notes: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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Summary: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.
Item Description:Gesehen am 10.06.2020
Physical Description:Online Resource
ISSN:1875-9777
DOI:10.1016/j.stem.2020.04.003

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