Effects of MIR-144/451 disruption on normal and β-thalassemic human erythropoiesis in vitro. CD34⁺ cells obtained from a healthy donor (HBB^+/+) or from a patient with β-thalassemia (HBB^thal) were electroporated with Cas9-sgRNA ribonucleoprotein complexes targeting the tandem MIR-144 and MIR-451 coding sequences then induced to undergo in vitro erythroid differentiation. (A) The tandem microRNA genes separated by 92 bp are shown on top. sgRNAs targeting MIR-144 and MIR-451 are shown below in red, and the protospacer adjacent motif (PAM) sequences are in blue. Sequence alignments showing the 4 most common indels detected via NGS 72 hours after editing are shown (bottom). Dashes indicate deletions. The size and frequency of each deletion are shown (right). (B) MIR-144/451 editing efficiency as measured via NGS of HSPCs. n = 3 for each genotype. (C) Western blot analysis of the indicated proteins in MIR-144/451–disrupted and control cells on day 12 of erythroid differentiation. (D) Results of multiple experiments performed as in panel C. CAB39 was normalized to β-actin, and pS6 was normalized to S6. Data from HBB^+/+ and HBB^Thal were normalized separately. (E) Representative flow cytometry plots showing the gating strategy for determining CD71 expression in developmental stage–matched erythroid precursors on day 12 of erythroid differentiation. (F) Summary of multiple experiments performed showing CD71 MFI in erythroblast populations defined by the gating strategy shown in panel E. (G) pThr172 AMPKα levels in erythroblasts on day 12 of differentiation as measured via sandwich enzyme-linked immunosorbent assay and normalized to total protein determined by the bicinchoninic acid assay (BCA). Bar charts show mean values ± SD of 3 biological replicate experiments using cells from the same CD34⁺ cell donors; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05.