Disruption of the BCL11A erythroid enhancer impairs human erythropoiesis in vivo. (A) CD34⁺ HSPCs were electroporated with RNPs targeting either BCL11A^E, CD33, or HBG^P and then transplanted separately into immunodeficient NBSGW mice (supplemental Figure 3A, left panel). At 16 weeks, recipient BM was isolated and analyzed by flow cytometry for the proportions of human myeloid (hCD11b⁺), B cell (hCD19⁺), and erythroid (hCD235⁺) lineages. Data are illustrated as mean ± SD. ∗∗∗∗FDR-adjusted P < .0001 determined by GEE models with post hoc comparisons. (B) Percentages of indels in flow cytometry-purified human donor-derived CD19⁺ B cells, CD11b⁺ myeloid cells, CD34⁺ lineage⁻ HSPCs, and CD235⁺ erythroid cells isolated from mice transplanted with BCL11A^E-, CD33-, or HBG^P-disrupted HSPCs. Each symbol datapoint represents an individual mouse transplanted with cells from a specific CD34⁺ HSPC donor represented by unique symbols. Data are illustrated as mean ± SD. ∗FDR-adjusted P < .05 or ∗∗P < .01 determined by paired t tests or exact Wilcoxon tests with post hoc comparisons. (C) CD34⁺ HSPCs were electroporated with RNPs targeting either BCL11A^E, CD33, or HBG^P. Equal numbers of cells targeted at 2 different loci were mixed and then transplanted into immunodeficient NBSGW mice (supplemental Figure 3A, right panel). At 16 weeks, indel frequencies were measured in human donor-derived CD19⁺ B cells, CD11b⁺ myeloid cells, CD34⁺ lineage⁻ HSPCs, and CD235⁺ erythroid cells purified from mouse recipient BM. Graphs reveal %indels (mean ± SEM) in mixtures of cells targeted at the indicated loci. ∗∗∗∗FDR-adjusted P < .0001 determined by paired t tests or exact Wilcoxon tests with post hoc comparisons. UT, untreated.