Genome-wide CRISPR knockout screen identifies endo-lysosomal trafficking complexes as regulators of prion cell-to-cell spread

The exact host factors governing the steps by which pathogenic prions spread from one cell to another and establish infection remain elusive. To visualise and quantify prion transfer from donor to recipient cells, we generated a human cell line (HovH) infectable with sheep prions and we differentiated between PrP^Sc and PrP^C using conformation-specific antibodies. Then, we performed a genome-wide knockout screen for modifiers of this process in human cells. Cas9-expressing naive cells were infected with a pooled CRISPR knockout library (t.spiezzo) and co-cultured with chronically prion-infected cells. At different time points, prion levels in the newly infected cells were quantified using specific antibodies. Cells with high or low fluorescence, corresponding to high or low levels of PrP^Sc, were sorted and sequenced to identify the gRNAs of genes enriched or depleted in these populations. Among the expected results, we identified several genes that are involved in the GPI-anchor biosynthesis and ER chaperones known to bind PrP^C protein (PDIA6, PDIA4, P4HB). Several components of the endo-lysosomal pathway emerge implicating multivesicular bodies -alongside early and recycling endosomes- as central sites for prion conversion. Notably, the Commander/Retriever sorting complex and the HOPS–Mon1/CCZ1 tethering module were essential for cell-to-cell spread. Future work will dissect how these trafficking complexes facilitate prion spreading and test their role across additional cell lines, different prion strains and organotypic mouse brain slices to identify if this is a universal mechanism for prion spread.