Project summary Mammalian prion diseases, such as Creutzfeldt-Jakob disease (CJD), Chronic Wasting Disease (CWD), bovine spongiform encephalopathy (BSE), and scrapie, are a group of infectious neurodegenerative disorders caused by the autocatalytic conversion of the host-encoded prion protein, PrPC, into a group of misfolded, infectious conformers collectively termed PrPSc. Multiple lines of evidence from biochemical, genetic, and cell biological studies suggest that PrPC and PrPSc employ specific pathways for biosynthesis, trafficking, and degradation in living cells. However, full elucidation of these pathways has been hindered the lack of tractable model organisms for genetic screening. To overcome this obstacle, we recently developed methods to detect both PrPC and PrPSc in brain-derived CAD5 cells using fluorescence-activated cell sorting (FACS). We will use these sensitive sorting methods to perform CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 whole genome library screens in CAD5 cells infected with different prion strains. These studies will be the first to fully map the molecular pathways that control the biosynthesis, trafficking, and degradation of both PrPC and PrPSc molecules, and thereby greatly advance our fundamental knowledge of prion cell biology. Our unbiased screens will also reveal the rate limiting steps of prion formation and clearance, thereby identifying the most susceptible targets for drug therapy.