CRISPR screens to identify and characterize ligands for glycan-binding proteins

Cell surface glycans regulate key biological processes including immune signaling, cell communication, and pathogen recognition. Glycan-driven signaling is primarily mediated by glycan-binding proteins (lectins), whose functions depend on the identity and presentation of their glycoprotein ligands. However, identifying ligands for glycan-binding proteins remains challenging due to the structural complexity of carbohydrates and the importance of cellular context in determining binding specificity. Here, we describe a fluorescence-activated cell sorting (FACS)-based pooled CRISPR screening workflow for systematic identification of genetic factors that regulate lectin binding in living cells. The protocol covers lentiviral transduction of pooled sgRNA libraries and phenotypic selection of high- and low-lectin-binding populations by flow cytometry. Genomic DNA extraction, sequencing, and computational sgRNA enrichment analysis enable identification of genes influencing ligand biosynthesis and presentation. Subsequent analysis of these genetic factors can provide a comprehensive view of the structural determinants that govern lectin-glycan binding. The approach is compatible with CRISPR knockout, interference, and activation strategies, allowing broad interrogation of both loss- and gain-of-function effects. Key considerations for maintaining library coverage, optimizing sorting parameters, and performing robust statistical analysis are highlighted to maximize screening performance. Overall, this workflow offers a scalable framework for mapping glycan ligand landscapes in health and disease.