Transient activation of CEBPA cisRE is sufficient to differentiate high-risk, stem cell–like AML cells. (A) qRT-PCR of CEBPA expression 3 days postelectroporation. Fold change represents ΔΔCt values compared to the sgNT condition. n = 3 independent replicates. Mean and SEM are shown. Two-sided Student t test was used for comparison. ∗∗P < .01. (B-D) Immunophenotypic analysis of a primary leukemia sample (patient 1, supplemental Table 9) 4 days postelectroporation. (B) Bivariate plot showing CD34 and CD117 expression assessed by flow cytometry. Black box denotes CD34⁺/CD117⁺ subset. (C) CD34 expression measured by MFI. (D) Percentage of CD34⁺/CD117⁺ cells. n = 3 independent replicates. Mean and SEM are shown. Two-sided Student t test was used for comparisons. ∗∗∗∗P < .0001. (E-F) Immunophenotypic analysis of a primary leukemia sample (patient 1, supplemental Table 9) 12 days postelectroporation. (E) Histogram showing CD11b expression assessed by flow cytometry. (F) Percentage of CD11b⁺ cells. n = 3 independent replicates. Mean and SEM are shown. Two-sided Student t test was used for comparison. ∗∗ P < .01. (G) Viable cell counts by trypan blue exclusion in primary leukemia sample (patient 1, supplemental Table 9) 8 days postelectroporation. n = 3 independent replicates. Mean and SEM are shown. Two-sided Student t test was used for comparison. ∗∗ P < .01. (H) qRT-PCR data of a panel of established HSC genes and LSC17 genes 18 days postelectroporation demonstrating the robust differentiation of a primary leukemia sample (patient 1, supplemental Table 9) following transient activation of CEBPA cisRE. n = 3 independent replicates. Mean and SEM are shown. Two-sided Student t test was used for comparison. ∗ P < .05. (I) Schematic of the experiment to assess the in vivo impact of CEBPA cisRE activation of a xenotransplanted primary leukemia sample (patient 1, supplemental Table 9). Cells were electroporated with mRNA encoding dCas9-VPR and 2 chemically synthesized sgRNAs targeting the CEBPA cisRE or a sgNT. Cells recovered in ex vivo culture for 2 days postelectroporation and then injected via the tail vein. All animals were euthanized 56 days after transplant for analysis of leukemia burden in spleens and BM. (J) Quantification of human cell chimerism (hCD45⁺) in the BM of mice transplanted with 1 × 10⁵ to 1 × 10⁶ cells and spleens of mice transplanted with 1 × 10⁶ cells. n = 4 to 10 xenotransplant recipients as shown. Mean and SEM are shown. Two-sided Student t test was used for comparison. ∗∗∗∗ P < .0001, ∗ P < .05. (K-L) Immunophenotypic analysis of the BM of mice transplanted with 1 × 10⁶ cells. Cells were labeled with a cocktail of antibodies including mouse CD45 and human CD45, CD34, CD117, and CD11b. (K) Bivariate plots depicting the gating strategy for quantification of engrafted leukemia stem/progenitor cells (CD34⁺/CD117⁺) and mature cells (CD11b⁺). Black boxes denote human cell subset (top), CD34⁺/CD117⁺ subset (middle), and CD11b⁺ subset (bottom). (L) Percentage of CD34⁺/CD117⁺ cells (top) and CD11b⁺ cells (bottom). n = 4 to 10 xenotransplant recipients as shown. Mean and SEM are shown. Two-sided Student t test was used for comparison. (M) Hematoxylin and eosin (H&E) staining of bone marrow of mice transplanted with 1 × 10⁶ cells. BM, bone marrow; MFI, mean fluorescence intensity, mRNA, messenger RNA; ns, not significant; sgNT, nontargeting sgRNA.