Genome-Scale CRISPR Screening in Human Intestinal Organoids Identifies Drivers of TGF-β Resistance

Till Ringel; Nina Frey; Femke Ringnalda; Sharan Janjuha; Sarah Cherkaoui; Stefan Butz; Sumana Srivatsa; Martin Pirkl; Giancarlo Russo; Lukas Villiger; Gerhard Rogler; Hans Clevers; Niko Beerenwinkel; Nicola Zamboni; Tuncay Baubec; Gerald Schwank

doi:10.1016/j.stem.2020.02.007

Genome-Scale CRISPR Screening in Human Intestinal Organoids Identifies Drivers of TGF-β Resistance

Till Ringel, Nina Frey, Femke Ringnalda, Sharan Janjuha, Sarah Cherkaoui, Stefan Butz, Sumana Srivatsa, Martin Pirkl, Giancarlo Russo, Lukas Villiger, Gerhard Rogler, Hans Clevers, Niko Beerenwinkel, Nicola Zamboni, Tuncay Baubec, Gerald Schwank

Hubrecht Institute

Onderzoeksoutput: Bijdrage aan wetenschappelijk tijdschrift/periodieke uitgave › Artikel › Wetenschappelijk › peer review

109 Citaten (Scopus)

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Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.

Originele taal-2	Engels
Pagina's (van-tot)	431-440.e8
Tijdschrift	Cell Stem Cell
Volume	26
Nummer van het tijdschrift	3
DOI's	https://doi.org/10.1016/j.stem.2020.02.007
Status	Gepubliceerd - 05 mrt. 2020

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10.1016/j.stem.2020.02.007

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Ringel, T., Frey, N., Ringnalda, F., Janjuha, S., Cherkaoui, S., Butz, S., Srivatsa, S., Pirkl, M., Russo, G., Villiger, L., Rogler, G., Clevers, H., Beerenwinkel, N., Zamboni, N., Baubec, T., & Schwank, G. (2020). Genome-Scale CRISPR Screening in Human Intestinal Organoids Identifies Drivers of TGF-β Resistance. Cell Stem Cell, 26(3), 431-440.e8. https://doi.org/10.1016/j.stem.2020.02.007

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title = "Genome-Scale CRISPR Screening in Human Intestinal Organoids Identifies Drivers of TGF-β Resistance",

abstract = "Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.",

author = "Till Ringel and Nina Frey and Femke Ringnalda and Sharan Janjuha and Sarah Cherkaoui and Stefan Butz and Sumana Srivatsa and Martin Pirkl and Giancarlo Russo and Lukas Villiger and Gerhard Rogler and Hans Clevers and Niko Beerenwinkel and Nicola Zamboni and Tuncay Baubec and Gerald Schwank",

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Ringel, T, Frey, N, Ringnalda, F, Janjuha, S, Cherkaoui, S, Butz, S, Srivatsa, S, Pirkl, M, Russo, G, Villiger, L, Rogler, G, Clevers, H, Beerenwinkel, N, Zamboni, N, Baubec, T & Schwank, G 2020, 'Genome-Scale CRISPR Screening in Human Intestinal Organoids Identifies Drivers of TGF-β Resistance', Cell Stem Cell, vol. 26, nr. 3, blz. 431-440.e8. https://doi.org/10.1016/j.stem.2020.02.007

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T1 - Genome-Scale CRISPR Screening in Human Intestinal Organoids Identifies Drivers of TGF-β Resistance

AU - Ringel, Till

AU - Frey, Nina

AU - Ringnalda, Femke

AU - Janjuha, Sharan

AU - Cherkaoui, Sarah

AU - Butz, Stefan

AU - Srivatsa, Sumana

AU - Pirkl, Martin

AU - Russo, Giancarlo

AU - Villiger, Lukas

AU - Rogler, Gerhard

AU - Clevers, Hans

AU - Beerenwinkel, Niko

AU - Zamboni, Nicola

AU - Baubec, Tuncay

AU - Schwank, Gerald

PY - 2020/3/5

Y1 - 2020/3/5

N2 - Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.

AB - Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.

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Genome-Scale CRISPR Screening in Human Intestinal Organoids Identifies Drivers of TGF-β Resistance

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