Abstract

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.

中文翻译：

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一个 GlycoGene CRISPR-Cas9 慢病毒文库，用于研究凝集素结合和人类聚糖生物合成途径

摘要

细胞表面蛋白质和脂质上的聚糖生物合成由不同类别的酶和蛋白质协调，包括： i．添加糖类的糖基转移酶；二. 修剪聚糖的糖苷酶；三. 调节细胞内运输的保守寡聚高尔基复合体成员；四. 帮助糖核苷酸生物合成的酶；和诉磺基转移酶。这份手稿描述了一个汇集的“glycoGene CRISPR”慢病毒文库，该文库针对参与上述过程的 347 个人类基因。每个糖原包含大约 10 个单向导 RNA (sgRNA)，假定的编辑位点跨越目标的长度。为了确定调节生物过程的糖基化途径，提出了数据分析方案。作为原理证明，呈现正向遗传筛选结果以鉴定调节 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、野豌豆凝集素、花生凝集素）与 HL 结合的穿透性糖原-60 个早幼粒细胞。除了验证先前建立的生物学之外，该研究还确定了三种酶 PAPSS1、SLC35B2 和 TPST2，它们是调节白细胞中主要 P-选择素糖蛋白配体 1 硫酸化的关键分子。本研究中使用的大约 80-90% 的 sgRNA 显示出高编辑效率，CRISPR 文库提取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 glycoGene CRISPR 文库包含高效 sgRNA。进一步，