MKO mediated by CRISPR/Cas9 reduces proliferation and clonogenicity of B-ALL cells in vitro and tumorigenicity in vivo. (A) Representative western blot with anti-MondoA antibody of Nalm6 MKO clone 1 and clone 2 lysates; Cas9-only transfected Nalm6 B-ALL cell line as control; GAPDH as loading control. (B) Long-term proliferation measured with BrdU assay (n = 6 for each time point, P < .01 at 48 hours and P < .001 at 72 hours, 2-way ANOVA). (C) Long-term proliferation measured by direct cell counting. Cas9-only transfected Nalm6 cell line and MKO clones 1 and 2, were grown under normoxia (20% O₂) (n = 6 for each time point, P < .001 at 72 hours and 96 hours, 2-way ANOVA). (D) Contact-independent growth of Nalm6 cells with CRISPR/Cas9-mediated MKO. Quantitative evaluation in methylcellulose-based colony formation assay (n = 4, P < .001, results are presented as mean plus or minus SD; 1-way ANOVA). (E) Survival curves of mice transplanted with Nalm6 MKO and Cas9-only control cells (n = 8 for MKO group, n = 9 for CTRL, P < .001). (F-G) Xenograft transplantation of Nalm6 cells with either MKO (n = 8) or control (n = 9) in Rag2^−/−yc^−/− mice. Weight of liver and spleens in grams (P = .0026 [liver] and P = .0008 [spleen], results are presented as mean plus or minus SD; unpaired Student t test). **P < .01; ***P < .001.