Categorization of screen hits based on effects on different dynein cargoes. (A) Grouping of hits based on effects in the secondary screen on localization of peroxisomes (PEX; average of GFP-BICD2N-FRB and PTS-RFP-FKBP), early endosomes (EEA1), and trans-Golgi network (TGN46). Cargo localization ratio was calculated by dividing the spot number in the perinuclear region by the spot number in the peripheral region (negative values indicate increased dispersion). Grouping was performed with K-means clustering with Euclidean distance. Data points represent the average rZ (central reference = NTC) from two independent experiments (at least four wells per crRNA per experiment), with each line representing a crRNA pool. Dashed lines show ± 2.5*SD of NTC. Individual clusters are labeled with the number of constituent genes (parentheses), examples of constituent genes, and manual annotation of predominant cargo signature. See Table S5 for the list of genes in each cluster. (B) Representative images of cargo localization in cells edited with crRNAs targeting known components and well-characterized interactors of the dynein machinery, as well as novel hits. Third column from the left is a merge of GFP-BICD2N-FRB and PTS-RFP-FKBP signals. Scale bar, 20 µm.