Genome-wide screens identify core regulators of cell surface prion protein expression

Expression of the cellular prion protein, PrP^C, on the surface of neurons plays an important role in the pathogenesis of prion disease. We performed genome-wide CRISPR/Cas9 knockout screens in prion-infectible cells of neuronal origin (CAD5) to identify regulators of cell surface PrP^C expression. We identified and validated 46 positive and 21 negative regulators of cell surface PrP^C expression in undifferentiated CAD5 cells. Pathway analysis of the screening dataset showed that genes involved in the glycophosphatidylinositol (GPI) anchor and N-glycosylation biosynthetic pathways were overrepresented as positive regulators of cell surface PrP^C. We also sought to determine whether the same or different genes regulate cell surface PrP^C in CAD5 cells that have been differentiated to a more neuronal state and validated 41 positive and 13 negative regulators of CAD5 cell surface PrP^C expression in the differentiated state. We identified 23 core genes as shared between the undifferentiated and differentiated cell states, including many positive regulators involved in GPI anchor biosynthesis. Intriguingly, unique regulators were also identified in the undifferentiated and differentiated cell states, suggesting that some mechanisms regulating cell surface PrP^C expression in CAD5 cells are dependent on cell state. This list of core genes involved in regulating cell surface PrP^C expression in a prion-susceptible, neuron-like cell type offers a valuable guide for future research and may help identify potential therapeutic targets for prion disease and other neurodegenerative diseases.