Chemical and Biological Engineering, Biomedical Engineering and Medicine, University at Buffalo, State University of New York, Buffalo, NY 14260, USA.
Glycobiology. 2021 Apr 1;31(3):173-180. doi: 10.1093/glycob/cwaa074.
Glycan biosynthesis on cell surface proteins and lipids is orchestrated by different classes of enzymes and proteins including the following: i. glycosyltransferases that add saccharides; ii. glycosidases that trim glycans; iii. conserved oligomeric golgi complex members that regulate intracellular transport; iv. enzymes aiding the biosynthesis of sugar-nucleotides; and v. sulfotransferases. This manuscript describes a pooled "glycoGene CRISPR" lentiviral library that targets 347 human genes involved in the above processes. Approximately 10 single-guide RNA (sgRNA) are included against each glycogene, with the putative editing site spanning the length of the target. A data analysis scheme is presented in order to determine glycosylation pathways regulating biological processes. As proof of principle, forward genetic screen results are presented to identify penetrating glycogenes that regulate the binding of P-/E-selectin, anti-sialyl Lewis-X monoclonal antibody HECA-452 and selected lectins (phaseolus vulgaris leucoagglutinin, vicia villosa lectin, peanut agglutinin) to HL-60 promyelocytic cells. Besides validating previously established biology, the study identifies three enzymes, PAPSS1, SLC35B2 and TPST2, as key molecules regulating sulfation of the major P-selectin glycoprotein ligand-1 in leukocytes. Approximately 80-90% of the sgRNA used in this study displayed high editing efficiency, and the CRISPR library picked up entire gene sets regulating specific biosynthetic pathways rather than only isolated genes. These data suggest that the glycoGene CRISPR library contains high-efficiency sgRNA. Further, this resource could be useful for the rapid screening of glycosylation-related genes and pathways that control lectin recognition in a variety of contexts.
细胞表面蛋白和脂质上的聚糖生物合成由不同类别的酶和蛋白质来调控，包括以下几种：i. 糖基转移酶，负责添加糖基；ii. 糖苷酶，负责修剪聚糖；iii. 保守的寡聚高尔基体复合物成员，调节细胞内运输；iv. 辅助糖核苷酸生物合成的酶；v. 硫酸转移酶。本文描述了一种汇集的“聚糖基因 CRISPR”慢病毒文库，该文库针对上述过程中涉及的 347 个人类基因。每个糖基因大约包含 10 个单引导 RNA(sgRNA)，潜在的编辑位点跨越靶标长度。本文提出了一种数据分析方案，以确定调控生物过程的糖基化途径。作为原理验证，本文呈现了正向遗传筛选结果，以鉴定穿透性糖基因，这些基因调节 P-/E-选择素、抗唾液酸 Lewis-X 单克隆抗体 HECA-452 和选定的凝集素（菜豆白细胞凝集素、菜豆植物血凝素、花生凝集素）与 HL-60 早幼粒细胞的结合。除了验证先前建立的生物学，该研究还鉴定了三种酶，即 PAPSS1、SLC35B2 和 TPST2，作为调节白细胞中主要 P-选择素糖蛋白配体-1 硫酸化的关键分子。本研究中使用的约 80-90%的 sgRNA 显示出高编辑效率，并且 CRISPR 文库捕捉到了调节特定生物合成途径的整个基因集，而不仅仅是孤立的基因。这些数据表明，聚糖基因 CRISPR 文库包含高效 sgRNA。此外，该资源可用于快速筛选糖基化相关基因和途径，以控制各种情况下凝集素的识别。
