CEBPA cisRE is necessary for differentiation of MECOM-driven AML cells. (A) Primary MECOM⁺ AML cells were harvested from patients at diagnosis and cryopreserved (supplemental Table 9). Cells were thawed for short-term ex vivo culture and electroporated with CRISPR-Cas9 RNPs to induce genetic perturbations at the MECOM vs AAVS1 locus ± CEBPA +42 kb cisRE. (B) Efficiency of gene editing in 3 biologically distinct primary AMLs at the AAVS1, MECOM, and CEBPA (cisRE) loci. Editing estimated using Sanger sequencing of amplicons followed by sequence trace decomposition analysis with Inference of CRISPR Edits (ICE) tool.⁵⁸ For CEBPA cisRE, only deletions resulting from dual guide cleavage were counted. n = 3 technical replicates. Mean and SEM are shown. (C-D) qRT-PCR of CEBPA and MECOM expression in all conditions 3 days postelectroporation. Fold change represents ΔΔCt values compared to the sgNT condition. n = 3 technical replicates, and mean and SEM are shown. n = 3 independent replicates, and mean and SEM are shown. Two-sided Student t test was used for comparison. ∗P < .05. (E-G) Immunophenotypic analysis of a primary leukemia sample (patient 1, supplemental Table 9) 6 days postelectroporation. (E) Bivariate plot showing CD34 and CD117 expression assessed by flow cytometry. Black box denotes CD34⁺/CD117⁺ subset. (F) Percentage of CD34⁺/CD117⁺ cells. (G) CD34 expression measured by MFI. n = 3 independent technical replicates. Mean and SEM are shown. A Mann-Whitney test was used for comparisons. ∗P < .05. (H-I) Immunophenotypic analysis of a primary leukemia sample (patient 2, supplemental Table 9) 8 days postelectroporation. (H) Histogram showing CD34 expression assessed by flow cytometry. (I) Percentage of CD34⁺ cells. n = 3 independent technical replicates. Mean and SEM are shown. A Mann-Whitney test was used for comparisons. ∗P < .05. (J-K) Immunophenotypic analysis of primary leukemia (patient 3, supplemental Table 9) 8 days postelectroporation. (J) Histogram showing CD34 expression assessed by flow cytometry. (K) Percentage of CD34⁺ cells. n = 3 independent replicates. Mean and SEM are shown. Two-sided Student t test was used for comparisons. ∗P < .05. MFI, mean fluorescence intensity; sgNT, nontargeting sgRNA.