A CRISPR/Cas9-Mediated FACS Screen Reveals Novel Regulators of Wnt/β-catenin Signalling in Human Embryonic Stem Cells

An important signalling mechanism in stem cell fate determination is the Wnt/β-catenin signalling pathway, which is critical in self-renewal and determination of multiple cell lineages. However, the underlying mechanisms involved in regulating Wnt signalling during self-renewal and differentiation of human embryonic stem cells (ESCs) are not fully understood. This thesis aims to identify and characterize regulators of Wnt/β-catenin signalling in hESCs using a CRISPR-Cas9 genome-wide screening approach. A fluorescent GFP reporter was knocked into the AXIN2 locus of a human ESC line containing an inducible Cas9, to enable screening using flow cytometry for regulators of AXIN2 expression. Validating our approach, several known regulators of Wnt/β-catenin signalling were identified. Novel regulators were also identified as negative regulators of AXIN2 expression, including METTL3 and other genes involved in the N6-methyladenosine RNA modification. While the mechanism remains to be elucidated, this study uncovers a novel link between m6A and Wnt/β-catenin signalling in hESCs.M.Sc.2021-11-20 00:00:0