Genome-wide KO gene screening identifies MYO18B as a key regulator of lysosomal exocytosis. (A) Schematic diagram of CRISPR/Cas9-based KO gene screening. After the GeCKO library was introduced to cells, cells showing low surface levels of LAMP1 were sorted. sgRNA sequences from sorted and unsorted populations were analyzed by deep sequencing. (B) Flow cytometry of THP-1 cells before and after three rounds of sorting for low cell surface LAMP1. (C) Scatter plot represents the gene screening results. The y-axis (beta score) is evaluated by the MAGeCK maximum likelihood estimation (MLE) algorithm, which describes the differentiated sgRNA reads between sorted and unsorted cells. (D) Flow cytometry of cell surface LAMP1 levels in WT and MYO18B-KO HeLa cell lines. See also Fig. S1 B. (E) Quantitative flow cytometry of cell surface LAMP1. Data are the relative mean ± SD from three independent experiments (One-way ANOVA with Dunnett correction; **P ≤ 0.01; ns, P > 0.05). (F) HEX secretion assay in HeLa cells. Data are the relative mean ± SD from three independent experiments (One-way ANOVA with Dunnett correction; ****P ≤ 0.0001; ***P ≤ 0.001). See also Fig. S1, E and F. (G) Schematic diagram of plasma membrane repairing assay mediated by SLO. (H) Plasma membrane repairing assay in HeLa cells. Rescue means MYO18B-KO cells stably expressing EGFP-MYO18B. See also Fig. S1 G. (I) Representative CLSM images of WT and MYO18B-KO HeLa cells stained by Mannitou Ab under unpermeabilized conditions. Bars, 10 µm. (J) Quantitative result of Mannitou Ab staining in WT and MYO18B-KO HeLa cells (mean ± SD; n = 15 images; N = 3 biological replicates; unpaired two-tailed t test; ****P ≤ 0.0001).