CRISPR/Cas9 and glycomics tools for glycobiology.

Infection with the protozoan parasite is a major health risk owing to birth defects, its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Unlike most eukaryotes, propagates in intracellular parasitophorous vacuoles, but like nearly all other eukaryotes, glycosylates many cellular proteins and lipids and assembles polysaccharides. glycans resemble those of other eukaryotes, but species-specific variations have prohibited deeper investigations into their roles in parasite biology and virulence. The genome encodes a suite of likely glycogenes expected to assemble -glycans, -glycans, a -glycan, GPI-anchors, and polysaccharides, along with their precursors and membrane transporters. To investigate the roles of specific glycans in , here we coupled genetic and glycomics approaches to map the connections between 67 glycogenes, their enzyme products, the glycans to which they contribute, and cellular functions. We applied a double-CRISPR/Cas9 strategy, in which two guide RNAs promote replacement of a candidate gene with a resistance gene; adapted MS-based glycomics workflows to test for effects on glycan formation; and infected fibroblast monolayers to assess cellular effects. By editing 17 glycogenes, we discovered novel Glc-Man-GlcNAc-type -glycans, a novel HexNAc-GalNAc-mucin-type -glycan, and Tn-antigen; identified the glycosyltransferases for assembling novel nuclear -Fuc-type and cell surface Glc-Fuc-type -glycans; and showed that they are important for growth. The guide sequences, editing constructs, and mutant strains are freely available to researchers to investigate the roles of glycans in their favorite biological processes.