Genome-wide arrayed CRISPR activation screen for prion protein modulators

Abstract The cellular prion protein (PrP C ) is an essential substrate for prion propagation, and its abundance strongly influences susceptibility to prion disease. To systematically identify genetic regulators of PrP C 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 PrP C levels across 22,442 individual genetic perturbations. This screen identified 531 genes whose activation significantly modulated PrP C 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 PrP C homeostasis and enables reuse for investigations of membrane protein trafficking, proteostasis, and prion biology.