Figure 4. CRISPR validation experiments confirm top PC9 synthetic lethal interactions.
(A) Boxplots of growth phenotypes for PC9-Cas9-mCherry cells expressing the indicated pgRNA compared to PC9-Cas9-GFP cells expressing a double-safe-targeting control pgRNA. Boxes indicate mean ± SEM of six biological replicates, which are shown as overlaid points. Growth phenotype is defined as the log2-scaled ratio of mCherry:GFP cell counts at the late time point compared to the day 1 mCherry:GFP cell counts. Expected DKO phenotypes are the sum of single KO growth phenotypes. The expected and observed DKO phenotypes were compared using a one-tailed t test. Data shown are for the time point with the most extreme difference between expected and observed DKO growth phenotypes, termed the late time point: CCNL1/CCNL2 (day 12), CDK4/CDK6 (day 7), MEK1/MEK2 (day 11), and OXSR1/STK39 (day 10). Full time course data are shown in Figure S3.
(B) Fluorescence microscopy images of competitive fitness assays on early (day 1) and late time points as indicated above for (A). Scale bar, 100 μM.
(C) Western blot validation of single KO and DKO pgRNA-induced gene inactivation. For CCNL1, pie charts of percent mutant alleles based on next-generation sequencing are shown due to lack of a suitable CCNL1 antibody for western blotting. Additional genomic DNA-level validation data are presented in Figure S4.
See also Figures S3 and S4 and Table S5.