Over 200 glycosyltransferases are involved in the orchestration of the biosynthesis of the human glycome , which is comprised of all glycan structures found on different glycoconjugates in cells. The glycome is vast, and despite advancements in analytic strategies it continues to be difficult to decipher biological roles of glycans with respect to specific glycan structures, type of glycoconjugate, particular glycoproteins, and distinct glycosites on proteins. In contrast to this, the number of glycosyltransferase genes involved in the biosynthesis of the human glycome is manageable, and the biosynthetic roles of most of these enzymes are defined or can be predicted with reasonable confidence. Thus, with the availability of the facile CRISPR/Cas9 gene editing tool it now seems easier to approach investigation of the functions of the glycome through genetic dissection of biosynthetic pathways, rather than by direct glycan analysis. However, obstacles still remain with design and validation of efficient gene targeting constructs, as well as with the interpretation of results from gene targeting and the translation of gene function to glycan structures. This is especially true for glycosylation steps covered by isoenzyme gene families. Here, we present a library of validated high-efficiency gRNA designs suitable for individual and combinatorial targeting of the human glycosyltransferase genome together with a global view of the predicted functions of human glycosyltransferases to facilitate and guide gene targeting strategies in studies of the human glycome.

中文翻译：

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用于 CRISPR/Cas9 靶向人类糖基转移酶基因组的经过验证的 gRNA 文库


超过 200 种糖基转移酶参与人类糖组生物合成的协调，该糖组由细胞中不同糖缀合物上发现的所有聚糖结构组成。糖组是巨大的，尽管分析策略取得了进步，但仍然很难破译聚糖在特定聚糖结构、糖缀合物类型、特定糖蛋白和蛋白质上不同糖位点方面的生物学作用。与此相反，参与人类糖组生物合成的糖基转移酶基因的数量是可控的，并且大多数这些酶的生物合成作用已被定义或可以合理的置信度进行预测。因此，随着简便的 CRISPR/Cas9 基因编辑工具的出现，现在似乎更容易通过生物合成途径的遗传解剖来研究糖组的功能，而不是通过直接的聚糖分析。然而，有效基因打靶构建体的设计和验证、基因打靶结果的解释以及基因功能向聚糖结构的翻译仍然存在障碍。对于同工酶基因家族涵盖的糖基化步骤尤其如此。在这里，我们提出了一个经过验证的高效 gRNA 设计库，适用于人类糖基转移酶基因组的个体和组合靶向，以及人类糖基转移酶的预测功能的全局视图，以促进和指导人类糖组研究中的基因靶向策略。