Using CRISPR/Cas9 to Identify Gene Interactions with Hexosamine Biosynthesis and N-Glycan Remodeling Pathway Enzymes

Genetic studies by classical mutagenesis and screening methods have revealed many molecular interactions and regulatory relationships in animal models. The hexosamine biosynthesis pathway and N-glycosylation are upregulated in most cancers and have shown to play a role in many cancer cell phenotypes. In this thesis, a CRISPR/Cas9 genome-wide targeted mutagenesis approach was employed to identify gene interactions with chosen genes-of-interest from these two pathways: NAGK, GFPT1, MGAT1 and MGAT5. The gene interactions identified suggest relationships between our genes-of-interest and cell-cell adhesion, cytoskeleton, and folate and nucleotide metabolism. Further characterization of metabolite levels in the gene-of-interest knockout cells was done to help understand potential gene interactions from the screen. For example, metabolic imbalance in mutant cells likely indicates cell stress and reactive oxygen species, consistent with PRDX1, an antioxidant, being a suggested genetic interactor in multiple screens. These findings provide new insight on vulnerabilities and genomic redundancies in cancer cells.M.Sc