<title>ABSTRACT</title>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 <italic>in vivo</italic> stem cell-driven epithelial regeneration, providing an excellent <italic>ex vivo</italic> 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 <italic>Apc</italic>, we found that knock-out (KO) of <italic>Alk</italic>, <italic>Bclaf3</italic> or <italic>Prkra</italic> supports the Wnt independent self-renewal of gastric epithelial cells <italic>ex vivo</italic>. In adult mice, expression of these factors is predominantly restricted to non <italic>Lgr5</italic>-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.