Effect of mutating JMJD1C ZFD and Jumonji domain on MLL-AF9 leukemogenesis. (A-B) CFC assay after transduction of MLL-AF9-Cas9 cells with sgRNAs against ZFD (e12.3, e12.4) and Jumonji domain (e20.1, e21.1). (A) Colonies in methylcellulose culture (upper panels). Individual colony morphology (original magnification ×80; lower panels). (B) Colony counts 1 week after plating. (C) Proliferation assay of sorted GFP⁺ (marks MLL-AF9) TdTomato⁺ (marks sgRNA) MLL-AF9-Cas9 leukemia cells after sgRNA transduction. Data are mean ± standard deviation (SD) of 3 independent experiments. The P value was determined by 2-way analysis of variance (ANOVA). For multiple comparisons, P < .05 for day 7 and P < .0001 for day 9 between Renilla and all other sgRNAs, with the exception of Renilla vs e12.3 on day 7 (not statistically significant). (D) Apoptosis assay (left panel). Data are mean ± SD from 3 independent experiments. Representative flow plot; numbers within the upper left quadrant indicate average percentage (right panel). (E) Representative flow cytometry analysis of Mac1 of MLL-AF9-Cas9 leukemia cells 7 days after transduction (left panel). Mean fluorescence intensity (MFI; geometric mean) of Mac1 expression (right panel). Data are mean ± SD from 3 independent experiments. (F) Survival curves of secondary recipient mice that received MLL-AF9-Cas9 cells after transduction with respective sgRNAs. P = .040 overall, log-rank test. Pairwise log-rank test P values between Renilla and JMJD1C sgRNAs: P = .025 for e12.3; P = .137 for e12.4; P = .096 for e20.1, and P = .048 for e21.1. (G) Peripheral blood GFP⁺TdTomato⁺ cell percentage 8 weeks after transplantation. P = .05, multiple comparison, 1-way ANOVA. Renilla vs e12.3, P = .06; Renilla vs e12.4, e20.1, and e21.1, P = .08 . *P < .05, **P < .01, ***P < .001, unpaired 2-tailed Student t test.