Development of Genetic Screening Methods for Functional Characterization of Cell-Surface Glycans

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All living cells are coated with a dense layer of sugar molecules called glycans. Many glycans are attached to cell surface proteins and profoundly affect cell function through modulating protein stability, conformation and activity. Identifying the functions of individual glycans is essential to understanding how human cells grow, differentiate and interact with their external environment. The long-term goal of my research program is to systematically map the functions of cell-surface carbohydrate molecules in human cells. In order to study glycan function, individual glycans must be structurally characterized and then selectively removed from cellular proteins. The impact of glycan deletion on various aspects of cellular biology can then be determined. As hundreds of distinct glycan structures can be attached to thousands of different proteins, there is an urgent need for new high-throughput methods that can be applied to simultaneously study many glycans in a single experiment. To meet this need, we are applying a set of cutting-edge gene editing tools (CRISPR base editors) to precisely and rapidly modify cellular glycoproteins. These studies build on my strong research track record and demonstrated expertise with applying CRISPR-Cas9 functional genomics to the study of carbohydrate biology. In recent progress, we have: 1) developed a computational pipeline for design of sgRNAs that induce mutation of protein glycosylation sites 2) generated model cell lines expressing base editor enzymes and 3) performed proof-of-principle experiments that demonstrate successful mutagenesis of protein glycosylation sites in living cells. Building on this work, our short-term objectives are to develop new methods and resources that facilitate high-throughput, multiplexed characterization of glycan structure and function: (1) We will optimize a protocol (GlycoScan) for mutagenesis of protein glycosylation sites that can be applied to study how glycan deletion affects the activity of cell surface signalling receptors. (2) We will structurally characterize glycans attached to cell-surface signalling receptors using a new method (In Situ Epitope Tagging, or ISET) for rapid isolation of endogenous glycoproteins. (3) We will build a "glycome-wide" genetic screening platform for unbiased determination of glycan function at site-level resolution. Our unique, inter-disciplinary research program will train 12 HQP in glycoscience, cell biology, and CRISPR functional genetics. This program will benefit Canada by producing a new cohort of research scientists with training in cutting-edge functional genomic techniques that are becoming essential in both academic and industrial research. Our work will also produce new methods, technologies and databases that will widely benefit the Canadian cell biology research community. This program will thus catalyze new breakthroughs in understanding some of the most fundamental molecular processes that underlie cellular life.