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CRISPR-Based Screening in Three-Dimensional Organoid Cultures to Identify TGF-β Pathway Regulators

Part of the Methods in Molecular Biology book series (MIMB,volume 2488)

Abstract

The CRISPR/Cas technology has revolutionized forward genetic screening, and thereby facilitated genetic dissection of cellular processes and pathways. TGF-β signaling is a highly conserved cascade involved in development, regeneration, and diseases such as cancer. Even though many core components of the signaling cascade have already been described, several context-dependent pathway modulators remain unknown. To address this knowledge gap, we have recently developed a CRISPR screening approach for identifying TGF-β pathway regulators in three-dimensional organoid culture systems. Here, we provide a detailed protocol describing this approach in human intestinal organoids. With adaptations, this screening method could also be applied to other organoid types, and to other signaling cascades such as EGF or WNT signaling, thereby uncovering important mechanism in regeneration and disease.

Key words

  • CRISPR screening
  • Small intestinal organoids
  • TGF-β
  • Cancer

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Acknowledgements

We thank Till Ringel for development of the method and valuable discussion on the protocol. Also, we thank Patrik Simmler for proof-reading the manuscript. Nina Frey is supported by an ETH PhD grant.

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Correspondence to Gerald Schwank .

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Frey, N., Schwank, G. (2022). CRISPR-Based Screening in Three-Dimensional Organoid Cultures to Identify TGF-β Pathway Regulators. In: Zi, Z., Liu, X. (eds) TGF-Beta Signaling. Methods in Molecular Biology, vol 2488. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2277-3_8

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  • DOI: https://doi.org/10.1007/978-1-0716-2277-3_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2276-6

  • Online ISBN: 978-1-0716-2277-3

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