Trevisan, C.; Wang, H.; Bouris, V.; Mead, S. H.; Yin, J.-A.; Aguzzi, A.

The cellular prion protein (PrPC) is an essential substrate for prion propagation, and its abundance strongly influences susceptibility to prion disease. To systematically identify genetic regulators of PrPC abundance, we performed an arrayed genome-wide CRISPR activation (CRISPRa) screen targeting 19,839 human protein-coding genes in human glioblastoma cells. Using a quantitative time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay, we measured PrPC levels across 22,442 individual genetic perturbations. This screen identified 531 genes whose activation significantly modulated PrPC abundance. A curated subset of 50 candidates was subjected to validation using independent TR-FRET assays and Western blotting, confirming 45 (90%) of tested hits. All raw and processed screening data, metadata, and analysis code are publicly available. This dataset provides a comprehensive resource for studying genetic regulation of PrPC homeostasis and enables reuse for investigations of membrane protein trafficking, proteostasis, and prion biology.