Fig. 2: Screen validation by high-content microscopy.

A Experimental pipeline used for hits validation by high-content microscopy. RPE-1 cells stably expressing Cas9 nuclease and EGFP are transfected with individual synthetic sgRNAs in 96-well plates. After 3 days, the obtained polyclonal gene-edited cells are subjected to a 24 h α-syn-PFFs-A633 uptake assay before fixation, nuclei stain with Hoechst, and quantification by high-content microscopy. An EGFP mask allows quantification of cell area, to which PFFs content is normalized. Imaging was done using a CX7 high-content microscope and quantification with the HCS Studio Cell Analysis software. B Heatmap summarizing validation data. On a per-cell basis, the EGFP area and PFFs-A633/cell area ratio were measured, for each indicated gene, and for two sgRNAs per gene (a custom sgRNA and one from the tKOv3 library). The mean cell area and mean PFFs-A633/cell area were calculated and reported as percent of control sgRNAs targeting the AAVS1 locus (see color coding at the top right). n = 4-7 independent experiments. Genes symbols are sorted alphabetically, with putative hits enriched in the Low PFFs population first, followed by putative hits enriched in the High PFFs population. Statistical test: one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.