A GlycoGene CRISPR-Cas9 lentiviral library to study lectin binding and human glycan biosynthesis pathways

• DOI: 10.1093/glycob/cwaa074
• Publication Date: 2020-08-04T19:18:48Z
• Authors: Yuqi Zhu; Theodore Groth; Anju Kelkar; Yusen Zhou; Sriram Neelamegham
• Keywords: 0301 basic medicine; 03 medical and health sciences; Binding Sites; Glycosylation; Polysaccharides; Lectins; Humans; HL-60 Cells; CRISPR-Cas Systems; Gene Library; 3. Good health

Abstract Glycan biosynthesis on cell surface proteins and lipids is orchestrated by different classes of enzymes including the following: i. glycosyltransferases that add saccharides; ii. glycosidases trim glycans; iii. conserved oligomeric golgi complex members regulate intracellular transport; iv. aiding sugar–nucleotides; v. sulfotransferases. This manuscript describes a pooled “glycoGene CRISPR” lentiviral library targets 347 human genes involved in above processes. Approximately 10 single-guide RNA (sgRNA) are included against each glycogene, with putative editing site spanning length target. A data analysis scheme presented order to determine glycosylation pathways regulating biological As proof principle, forward genetic screen results identify penetrating glycogenes binding P-/E-selectin, anti-sialyl Lewis-X monoclonal antibody HECA-452 selected lectins (phaseolus vulgaris leucoagglutinin, vicia villosa lectin, peanut agglutinin) HL-60 promyelocytic cells. Besides validating previously established biology, study identifies three enzymes, PAPSS1, SLC35B2 TPST2, as key molecules sulfation major P-selectin glycoprotein ligand-1 leukocytes. 80–90% sgRNA used this displayed high efficiency, CRISPR picked up entire gene sets specific biosynthetic rather than only isolated genes. These suggest glycoGene contains high-efficiency sgRNA. Further, resource could be useful for rapid screening glycosylation-related control lectin recognition variety contexts.