Depletion of CAB39 eliminates the beneficial effects of miR-144/451 disruption in β-thalassemia. Lineage-negative (Lin⁻) E14.5 Hbb^th3/+miR^−/− fetal liver HSPCs (expressing CD45.2) were electroporated with Cas9 + Cab39 sgRNA ribonucleoprotein (RNP) complex (edited) or Cas9 only (mock), then transplanted into lethally irradiated C57BL/6 recipient mice (expressing CD45.1). (A) Cab39 editing efficiency, as measured via next-generation sequencing (NGS) of HSPCs maintained in vitro for 72 hours after electroporation (before transplantation) or of whole bone marrow cells collected from recipient mice 8 weeks after transplantation. n = between 3 and 6 mice for each condition. (B) Western blot analysis of CAB39 in Lin⁻ HSPCs maintained in vitro for 72 hours after electroporation. (C) Results of multiple experiments performed as in panel B. The y-axis represents the relative protein staining intensity on western blots as measured by automated image analysis, normalized to β-actin, and shown in arbitrary units. (D-G) Erythroid indices (y-axis) according to genotype (x-axis) at 8 weeks after transplantation. n = 7 mice for each condition. (H) Representative images of blood smears (Giemsa stain) from mice with the indicated genotypes. Images were obtained using an Eclipse Ni microscope and a 40× objective lens. Bars represent 5 μm. (I) Summary of spleen weights. n = 7 mice for each condition. (J) Flow cytometry quantification of S6 phosphorylation in maturation stage–matched bone marrow erythroblasts. n = 3 mice for each genotype. Bar charts show data as mean values ± SD; ∗∗∗∗P <.0001; ∗∗∗P <.001; ∗∗P <.01; ∗P < .05. RNP, ribonucleoprotein.