Disruption of the BCL11A erythroid enhancer impairs in vitro human erythropoiesis. Healthy human adult donor peripheral blood mobilized CD34⁺ HSPCs were electroporated with RNP complexes consisting of Cas9 + sgRNA targeting either the CD33 protein coding region, the BCL11A intron 2 erythroid enhancer (BCL11A^E), or the BCL11A binding motif in the γ-globin genes (HBG^P) grown in HSPC maintenance medium for 24 hours and then switched to erythroid differentiation medium. (A) Western blot revealing BCL11A-XL and β-actin protein levels on day 7 of erythroid differentiation. (B) Live cell numbers on days 5, 7, 13, and 18 of erythroid differentiation. Symbols illustrate the average normalized values for 3 different CD34⁺ HSPC donors. Data are illustrated as mean ± standard error of the mean (SEM). ∗∗∗FDR-adjusted P < .001 determined by generalized estimating equation (GEE) models with post hoc comparisons. (C) Apoptosis measured by flow cytometry at days 5 and 13 of erythroid differentiation. Graph reveals %annexin V⁺, propidium iodide positive or negative cells from replicate experiments, each with 2 different CD34⁺ HSPC donors. Data are illustrated as mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 indicate FDR-adjusted paired t tests or exact Wilcoxon tests with post hoc comparisons. (D) Equal numbers of cells targeted separately at 2 different loci (BCL11A^E, CD33, or HBG^P) were mixed and transferred to erythroid (Ery) or myeloid differentiation media. Graphs illustrate indel frequencies over time. The expected indel frequency at each target site was calculated as 50% of the indel frequency in cells targeted with each individual RNP (supplemental Figure 2B). Each symbol represents a separate experiment with unique donor CD34⁺ HSPCs. Data are illustrated as mean ± SD. ∗FDR-adjusted P < .05 determined by paired t tests or exact Wilcoxon tests with post hoc comparisons. (E) On days 4, 7, and 14 of erythroid differentiation, mixtures of cells targeted at different loci as described for panel D were fractionated into defined maturation stages by flow cytometry (supplemental Figure 2C) and indel frequencies of each targeted gene were determined. Erythroid precursors in order of increasing maturation stage are as follows: BFU-E, burst-forming unit erythroid; CFU-E, colony-forming unit erythroid; ProE, proerythroblast; BasoE, basophilic erythroblast; and PolyE, polychromatophilic erythroblast. Graphs show data from an experiment with duplicate using a healthy donor CD34⁺ HSPCs. UT, untreated.