CRISPR screens identify genes relevant for ruxolitinib sensitivity. (A) Schematic representation of the CRISPR-Cas9 screen in the presence or absence of ruxolitinib. MUTZ5 cells were treated with ruxolitinib 7 days after library transduction and cultured for 10 more days. sgRNA abundance before and after culture was assessed as described.¹⁸ (B) Dot graph representing β scores of 19 114 genes in the presence or absence of ruxolitinib. Genes whose β scores exhibited statistically significant change upon ruxolitinib treatment (FDR <0.25) are depicted in red (sgRNA more enriched with ruxolitinib) or blue (sgRNA more depleted with ruxolitinib). (C) Genes whose sgRNA abundance was significantly altered by ruxolitinib treatment were assessed using the DrugZ program.³² Genes that possess positive (C) or negative (D) z scores are plotted according to statistical values. (E) MUTZ5 cells were transduced with a lentivirus vector encoding a sgRNA and the Crimson cassette and cultured with or without 100 nM ruxolitinib, which is the ruxolitinib concentration used for the CRISPR screen shown in (A). Crimson-positive cell fractions were measured at indicated times by FACS. At each time point, that proportion was normalized to the number of Crimson-positive cells present at day 0 (2 days after transduction). Empty vector served as control. Each condition was assessed in triplicate, and data are represented as means ± SD. P values were calculated using an unpaired t test. (F) MUTZ5 clones stably expressing sgRNA against CRKL or FLT3 were established, treated with or without ruxolitinib (Ruxo) (1 μM, 2 hours) as described^43,50,58,65 and analyzed by western blotting.