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

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.

Keywords: CRISPR-Cas9; forward genetic screen; gene editing; glycoscience; selectin.

Fig. 1

Creation of GlycoGene CRISPR library transduced cells. A. Schematic of lentiviral vectors used in the CRISPR screen. dTomato acts as a surrogate reporter of Cas9 nuclease activity in SiC-V1-scr. GlycoGene CRISPR library was cloned in a doxycycline-inducible vector with BFP fluorescence reporter. B. An HL60-Cas9 cell line was established by transduction with SiC-V1-scr vector, and single cell flow sorting for isogenic clone expressing high levels of dTomato reporter (Bi–ii). A virus pool synthesized from the glycoGene CRISPR sgRNA library (U6-Dox-sgRNA) was applied to these cells. Two days later, 42% of the cells were BFP positive (Biii). One microgram per milliliter Dox was added starting at day 2 to initiate gene editing. Dox was removed and Cas9G7 sgRNA electroporated on day 4 to inactivate Cas9 activity. dTomato⁻BFP⁺ cells (arrow) were FACS sorted at day 12 in order to establish the stable HL-60 glycoGene cell library (called “HL60-lib”). Flow cytometry plots represent: i. wild-type HL60; ii. HL60-Cas9; iii. HL60-Cas9 with glycoGene library; iv. cells after Cas9 nuclease editing on day 6; and v. HL60-lib cells on day 20. dTomato fluorescence of HL60-lib on day 20 (Bv) is similar to wild-type HL60s (Bi) confirming complete knockdown of Cas9 activity. (This figure is available in black and white in print and in color at Glycobiology online)

Fig. 2

GlycoGene screening for altered lectin binding using MAGeCK. A. HL60 cells containing the full glycoGene library (i.e. HL60-lib, BFP⁺dTomato⁻) were sorted on day 12. On the same day, three color sorts were also performed to select for BFP⁺dTomato⁻ cells exhibiting low E-selectin IgG binding (A), low P-selectin IgG binding (B), low HECA-452/sialyl Lewis-X expression (C), low PHA-L binding (D), high VVA binding (E) and low PNA binding (F). Either one or two additional sorts were performed to obtain “lectin-selected” cell populations (left panels). Red histogram marks the binding of HL60-Cas9 control, gray histogram marks the negative control (isotype/blocking antibody) and black line marks the final lectin-selected population. sgRNA enriched in the lectin-selected population were identified using MAGeCK following deep sequencing (right panels). Genes identified with FDR < 10⁻³ are marked in the individual panels, next to the relevant data point. (This figure is available in black and white in print and in color at Glycobiology online)

Fig. 3

LFC analysis. Log₂-fold change sgRNA data are presented for target gene vs. nontarget control for the unselected HL60-lib cells, and also the six lectin-selected cell types, i.e. LFC = log₂[sgRNA count for target gene/sgRNA count for nontarget gene]. These data are presented for 20 different glycogenes that are involved in the following: A. CMP-sialic acid biosynthesis; B. GDP-fucose biosynthesis; C. α(1,3)fucosyltransferase FUT7 and α(2,3)sialyltransferase ST3Gal-4 activity; D.  O-glycan construction; E. protein tyrosine sulfation; and F.  N-glycan elaboration. Each dot represents one sgRNA. Enriched sgRNA display LFC > 0. Unaffected sgRNA have LFC ≤ 0, similar to the HL60-lib control. (This figure is available in black and white in print and in color at Glycobiology online)