A genome-scale CRISPR screen reveals factors regulating Wnt-dependent renewal of mouse gastric epithelial cells

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 renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that 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 self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3β, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.

A Platform for Validating Colorectal Cancer Driver Genes Using Mouse Organoids

“…The organoid culture has enabled us to assess the reponse of intestinal epithelial cells to anti-cancer drugs, growth factors, or inflammatory cytokines and analyze the detailed molecular mechanisms in vitro (Lindemans et al, 2015 ; De Sousa E Melo et al, 2017 ; Kajino-Sakamoto et al, 2021 ). A recent study has performed a genome-scale CRISPR screen to identify genes that regulate Wnt-dependent renewal of gastric epithelial cells and identified Alk, Bclaf3 , and Prkra as Wnt pathway regulators (Murakami et al, 2021 ); the data show that mouse organoids are also useful for stem cell research.…”

An organoid CRISPRi screen revealed that SOX9 primes human fetal lung tip progenitors to receive WNT and RTK signals

“…Large scale functional genomic studies via genome-wide screens in cancer cell lines have revealed previously unappreciated genetic regulators and accelerated the drug discovery process (Bowden et al, 2020;Doench et al, 2016;Sanjana et al, 2014). CRISPR screens have started to be applied to 3D organoid systems which can more faithfully recapitulate in vivo biological processes (Murakami et al, 2021;Planas-Paz et al, 2019;Ringel et al, 2020), but so far have required large cell numbers to overcome variability. CRISPR interference (CRISPRi) is particularly useful for the generation of homogenous knockdowns to study essential gene function (Gilbert et al, 2014;Mandegar et al, 2016).…”

A CRISPR knockout screen reveals new regulators of canonical Wnt signaling

“…It should be noted that different types of genome-wide screens (gene-trap, siRNA and CRISPR) aimed at the identification of new Wnt signaling components yield different hits [32,33]. Importantly, even the most known and robust Wnt signaling regulators were not highly scored in most cases and the top hits widely differ between individual screens [12,[32][33][34][35][36][37][38][39][40]. This observation was previously discussed [32] and it was suggested that distinct screening systems i.e., different libraries or cell lines used, differences in phenotypic scoring, the length of the screen and other factors affecting knockout efficiency influence the screen results.…”