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

所有的活细胞都被一层致密的糖分子包裹着，称为聚糖。许多聚糖附着在细胞表面蛋白质上，通过调节蛋白质的稳定性、构象和活性而深刻地影响细胞功能。确定单个聚糖的功能对于了解人类细胞如何生长，分化以及与外部环境相互作用至关重要。我的研究计划的长期目标是系统地绘制人类细胞中细胞表面碳水化合物分子的功能。为了研究聚糖的功能，必须对单个聚糖进行结构表征，然后选择性地从细胞蛋白质中去除。然后可以确定聚糖缺失对细胞生物学各个方面的影响。由于数百种不同的聚糖结构可以连接到数千种不同的蛋白质上，因此迫切需要新的高通量方法，可以在单个实验中同时研究许多聚糖。为了满足这一需求，我们正在应用一套尖端的基因编辑工具（CRISPR碱基编辑器）来精确快速地修饰细胞糖蛋白。这些研究建立在我强大的研究记录基础上，并展示了将CRISPR-Cas9功能基因组学应用于碳水化合物生物学研究的专业知识。在最近的进展中，我们已经：1）开发了用于设计诱导蛋白质糖基化位点突变的sgRNA的计算管道，2）生成了表达碱基编辑酶的模型细胞系，以及3）进行了证明活细胞中蛋白质糖基化位点的成功诱变的原理验证实验。在这项工作的基础上，我们的短期目标是开发新的方法和资源，促进高通量，多路复用的表征聚糖的结构和功能：（1）我们将优化的协议（GlycoScan）的蛋白质糖基化位点的诱变，可应用于研究如何聚糖删除影响细胞表面信号受体的活性。(2)我们将使用一种新的方法（原位表位标记，或ISET）快速分离内源性糖蛋白，从结构上表征附着在细胞表面信号受体上的聚糖。(3)我们将建立一个“糖组范围”的遗传筛查平台，用于在位点水平分辨率下无偏地确定糖功能。 我们独特的跨学科研究计划将在糖科学，细胞生物学和CRISPR功能遗传学方面培训12名HQP。该计划将通过培养一批新的研究科学家来使加拿大受益，这些科学家接受了在学术和工业研究中越来越重要的尖端功能基因组技术的培训。我们的工作还将产生新的方法，技术和数据库，这将使加拿大细胞生物学研究界广泛受益。因此，该计划将促进理解细胞生命基础的一些最基本的分子过程的新突破。

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.