Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids

• Published in: Cell stem cell Vol. 26; no. 5; pp. 782 - 792.e7
• Main Authors: Michels, Birgitta E., Mosa, Mohammed H., Streibl, Barbara I., Zhan, Tianzuo, Menche, Constantin, Abou-El-Ardat, Khalil, Darvishi, Tahmineh, Członka, Ewelina, Wagner, Sebastian, Winter, Jan, Medyouf, Hind, Boutros, Michael, Farin, Henner F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.05.2020
• Subjects: clonal drift; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; Colon; colorectal cancer; CRISPR-Cas Systems - genetics; Genes, Tumor Suppressor; human colonic stem cells; Humans; lentiviral barcoding; non-homologous end joining; Organoids; patient-derived organoids; pooled-barcoded CRISPR-Cas9 screening; tumor microenvironment; tumor suppressor genes; unique molecular identifiers; lentiviral barcoding; pooled-barcoded CRISPR-Cas9 screening; human colonic stem cells; tumor suppressor genes; colorectal cancer; unique molecular identifiers; patient-derived organoids; non-homologous end joining; tumor microenvironment; clonal drift
• ISSN: 1934-5909; 1875-9777
• DOI: 10.1016/j.stem.2020.04.003

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. [Display omitted] •An optimized protocol for pooled CRISPR-Cas9 library screening in human colon organoids•Organoid xenografts enable unbiased identification of in vivo tumor suppressors•gRNA functionality in organoids is less predictable compared to 2D cancer cell lines•Clonal tracing with a UMI library allows adjustment for clonal drift during selection To enable forward genetic screens in human epithelia, Michels et al. devised a pooled CRISPR-Cas9 library screening strategy in human colon organoids. This enabled identification of tumor suppressors in vitro and after transplantation in vivo and, when combined with unique molecular identifiers (UMIs), allowed phenotypic studies on a clonal level.