A genome-wide CRISPR/Cas9 screen reveals KDM2B as a modifier of protein binding to heparan sulfate

Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and are involved in many important biological processes. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N- and O-sulfated. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. While most of the enzymes involved in HS biosynthesis have been studied extensively, much less information exists regarding the specific mechanisms that give rise to the variable composition and binding properties of HS. We conducted genome-wide CRISPR/Cas9 screens in a human melanoma cell line (A375) using guanidinylated neomycin (GNeo), a low molecular weight HS-dependent ligand. We implemented two complimentary screening strategies where GNeo was either conjugated to a cytotoxin or a fluorescent probe, and we screened for cell populations with increased survival or reduced binding to HS, respectively. Top hits from the screens were characterized and categorized based on their predicted gene functions and were examined for their involvement in the regulation of HS composition. Among the resulting targets, we identified many well-known HS biosynthesis enzymes as well as candidate genes with a previously unrecognized link to HS biosynthesis. The top hit shared between both screens was KDM2B, a histone lysine demethylase, for which we found a novel regulatory role in HS biosynthesis.