Confirmation of cargo localization phenotypes with independent crRNAs. Heatmap displaying localization ratio of dynein cargoes at the perinuclear versus peripheral region of cells treated with indicated crRNAs. GFP-BICD2N-FRB and PTS-RFP-FKBP were evaluated in U-2 OS PEX cells treated with rapamycin, whereas other markers were evaluated in unmodified, untreated U-2 OS cells. crRNAs were synthesized based on the VBC score (labeled “V”), except for LIS1 and DYNC1H1 crRNAs from the initial “Horizon Discovery” set (labeled “D”), which were used as additional positive controls. Bold labeling indicates crRNAs that target novel constituents of the dynein–dynactin cluster previously generated by unsupervised profiling. Color scales of individual features were adjusted based on their minimum and maximum values. Categories of affected cargoes were manually annotated based on statistically significant effects (see Fig. S7). Data represent relative change of mean per cell values aggregated at well level compared with NTC from a minimum of three independent experiments (minimum of 100 cells analyzed per well; four wells analyzed per condition).