Figure 1.

Identification of SubAB Resistance Genes in a Genome-wide CRISPR Screen

(A) Identification of sgRNAs enriched in the screen. Fold enrichment represents the average of two independent experiments. Genes, including at least one sgRNA enriched in duplicate, are aligned in descending order of fold enrichment. Orange and green bars indicate that multiple sgRNAs were enriched for a gene, whereas blue bars indicate that a single sgRNA was enriched for a gene. The full raw dataset is shown in Data S1, S2, and S3.

(B) Reproducibility of SubAB resistance conferred by individual sgRNAs. Each sgRNA was transduced into HeLa cells. Untransfected cells were excluded using puromycin selection, and successfully transfected cells were then treated for 24 h with SubAB at the indicated concentration (boxes) and further cultured for 4 days in the absence of the toxin. Viability was estimated using an 3-(4,5-Dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and is expressed as the percentage of the MTT value (OD570) in the absence of SubAB. The percentages shown are the mean percentages ± SD obtained from four independent experiments. The dotted line indicates the viability of mock-transfected cells treated with 0.3 ng/mL SubAB. The Holm-Bonferroni corrected t test was used for multiple comparisons. Asterisks denote statistical significance.

(C) Fold enrichment of six sgRNAs in glycan-related and membrane-trafficking genes enriched in the SubAB screen compared with that of an STx screen (Yamaji et al., 2019). The heatmap is representative of individual sgRNA enrichment (sg1–6) in two independent experiments (groups #1 and #2).

See also Figures S1 and S2, and Data S1, S2, and S3.