Fig. 2

Genome-wide CRISPR/Cas9 screen identifies genes protective to αS seeding. A αS biosensor line stably expressing spCAS9 was transduced with sgRNA lentiviral library (Brunello). Following antibiotic selection, biosensors were seeded with αS PFF (10nM) and flow sorted 24 hours later. Genomic DNA from positive and negative groups as well as unsorted total population were collected and decoded by NGS. B Volcano plot of genes identified in the screen. Colored in blue are all the genes plotted from FRET- group, while colored in black are those plotted from FRET+. Red dots are protective hits from both groups (5% FDR), specifically genes with a fold change <0.5 that were underrepresented in FRET- cells and genes >2 that were overrepresented in the FRET+ cells. C Pathway analysis of 154 protective genes via g:profiler. The enriched pathway is visualized by cytoscape. D Normalized FRET from αS biosensors following siRNA knockdown of 9 genes identified in the screen and αS PFF treatment. (n ≥9 repeats ; *** p < 0.001, ** p < 0.01 and * p < 0.05 by one-way ANOVA. error bars are ± S.E.M.). E Immunoblot with anti-VCP and anti-GAPDH of cell lysates from αS biosensors/spCas9 cells treated with a VCP gRNAs at low (L) medium (M) and high (H) concentration and harvest at 6 days after transduction demonstrates VCP at 16.5% non-treated controls in the high concentration treatment. F Normalized FRET of αS biosensors/spCas9 cells treated with a VCP gRNA or scrambled control gRNA following 24-hour application of αS PFF. (p = 0.0439 by a two-tailed student’s t-test). n = 4 biologically independent FRET assay. Data presents as mean ± S.E.M