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bioRxiv · 10.1101/2020.07.17.208496

Genome-Scale CRISPR Screening reveals novel factors regulating Wnt-dependent regeneration of mouse gastric organoids

Murakami, K.·Terakado, Y.·Siato, K.·Jomen, Y.·Takeda, H.·Oshima, M.·Barker, N.
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Abstract

An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial regeneration, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a Genome-Scale CRISPR Knock-Out (GeCKO) screening assay using mouse gastric epithelial organoids to identify novel modulators of Wnt-driven stem cell-dependent epithelial regeneration in the gastric mucosa. In addition to known Wnt pathway regulators such as Apc, we found that knock-out (KO) of Alk, Bclaf3 or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing Wnt-dependent self-renewal of gastric epithelia. Furthermore, using comprehensive RNA-sequencing analysis, we found that these factors influence epithelial regeneration by regulating Wnt signalling, apoptosis and expression of Reg family genes which could contribute to the epithelial regeneration through JAK/STAT3 pathway.

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Murakami, K., Terakado, Y., Siato, K., Jomen, Y., Takeda, H., Oshima, M., Barker, N.. 2020-07-17. Genome-Scale CRISPR Screening reveals novel factors regulating Wnt-dependent regeneration of mouse gastric organoids. https://doi.org/10.1101/2020.07.17.208496

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