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Cited by 222CRISPR and Genetic EngineeringChromatin Remodeling and CancerCancer Genomics and Diagnostics

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

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Michels, Birgitta E., Mosa, Mohammed H., Streibl, Barbara I., Zhan, Tianzuo, Menche, Constantin, Abou-El-Ardat, Khalil, Darvishi, Tahmineh, Członka, Ewelina +5 more·2020·Cell Stem Cell
DOI 10.1016/j.stem.2020.04.003
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Abstract

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-/-;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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  • CRISPR cutting screens in 3D organoids have been implemented to explore factors such as growth factor dependency for cell growth, identify potential cancer-driving forces, and uncover key genetic events in cell differentiation.

    “Even more exciting is the implementation of CRISPR cutting screens in 3D organoids to explore factors such as growth factor dependency for cell growth14–16, identifying potential cancer-driving forces17–19, and uncovering key genetic events in cell differentiation20–22.”

    From Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions (2025) · cites this paper as [17]

  • CRISPR screening combined with organoids has revealed power for discovering new gene functions.

    “Recent studies combining CRISPR screening technology with organoids have revealed the power of such strategies for discovering new gene functions12,13.”

    From Single-cell brain organoid screening identifies developmental defects in autism (2023) · cites this paper as [13]

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References 53 · Cited by 222

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Authors
Michels, B. E., Mosa, M. H., Streibl, B. I., Zhan, T., Menche, C., Abou-El-Ardat, K., Darvishi, T., Członka, E., Wagner, S., Winter, J., Medyouf, H., Boutros, M., & Farin, H. F.
Affiliations
  • Goethe University Frankfurt (DE)
  • German Cancer Research Center (DE)
Corresponding author
Farin, Henner F.
ORCID
Venue
Cell Stem Cell
Publisher
Elsevier BV
Published
2020-05
Volume
26
Issue
5
Pages
782-792.e7
ISSN
  • 1875-9777 (ISSN-L)
  • 1934-5909
DOI
10.1016/j.stem.2020.04.003
OpenAlex
W3020093160
Cited by
222
References
53
Citations by year
2026: 26 · 2025: 41 · 2024: 37 · 2023: 33 · 2022: 33 · 2021: 38 · 2020: 14
FWCI
9.45
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell stem cell (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Deutsche Forschungsgemeinschaft
  • Hessisches Ministerium für Wissenschaft und Kunst (III L 5-519/03/03.001-(0015))
  • José Carreras Leukämie-Stiftung (DJCLS 04FN/2018)
  • Five Colleges
  • Deutsche Krebshilfe (70112869)
  • Deutschen Konsortium für Translationale Krebsforschung (SPP 2084)
  • European Research Council (639795)
  • Verband der Chemischen Industrie
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer Genomics and Diagnostics
Keywords
colorectal cancer, colon organoids, CRISPR-Cas9 screen, tumor suppressor, TGFBR2, TGF-beta resistance, APC, KRAS G12D, xenografts, unique molecular identifiers, guide RNA design, functional genomics
MeSH terms
Colon, Humans, Organoids, Genes, Tumor Suppressor, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems

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