CRISPR/Cas9-based kinome screen identify modifiers of ASNase sensitivity in BCP-ALL. (A) Schematic representation of our screening strategy. Nalm6 cells were transduced stepwise with a doxycycline-inducible Cas9 and a kinome sgRNA library. Cells were cultured for 2 weeks in the presence of 2 μg/mL of doxycycline to induce Cas9 expression and 1 week in the absence of doxycycline. Then, cells were treated for 2 weeks with 5 IU/mL of ASNase, DNA was isolated and subjected to massively parallel sequencing, and results were analyzed using the MAGeCK algorithm. (B) Gene list of expressed gRNA targets that significantly modulate ASNase response, ranked by P value calculated using the MAGeCK algorithm. (C) Counts of individual gRNAs targeting TRIB3, GCN2, and BTK, respectively, before and after ASNase treatment. (D,F) Immunoblot analysis of TRIB3 or GCN2 protein expression in cells upon CRISPR/Cas9-based targeting of GCN2 or TRIB3, respectively. (E) ASNase-induced cell death as determined by quantification of cells positive for amine-reactive dyes using flow cytometry in Nalm6 WT and Nalm6 GCN2-deleted cells after a 3-day treatment with 1 IU/mL of ASNase. Each bar represents a mean of 3 independent experiments. ***P < .001; **P < .01; ***P < .05 (2-tailed, unpaired Student t test). (G) ASNase-induced cell death as determined by quantification of cells in subG₁ phase using flow cytometry of Hoechst-stained cells. Bars represent mean ± standard error of the mean (SEM) of n = 3 independent experiments. ***P < .001; **P < .01; ***P < .05 (2-tailed, unpaired Student t test). (H) Cell viability as measured by MTT in Nalm6 WT, and TRIB3del cells after treatment with the indicated dose of ASNase. Bars represent mean ± SEM of n = 3 independent experiments. ***P < .001; **P < .01; ***P < .05 (2-tailed, unpaired Student t test).