Extended Data Fig. 2: Co-depletion of NFIA and NFIX reactivates γ-globin in primary erythroblasts derived from healthy donors.
From: Dual function NFI factors control fetal hemoglobin silencing in adult erythroid cells
a, Three-phase in vitro culture system for human CD34+ HSPCs to differentiate into mature red cells. Cas9 and indicated NFI sgRNA RNPs were transfected into CD34+ HSPCs on day 4 of culture by electroporation. Erythroblasts were harvested and analyzed by RT-qPCR, immunoblot, HbF staining, and HPLC on day 13 or 15 of culture. b, Representative immunoblots of NFIA, NFIX, and γ-globin from untargeted primary cells at indicated days of differentiation. Experiments were performed on two donors with similar results. c, Representative HbF staining result from indicated gene edited primary erythroblasts at day 15 of differentiation. d, Representative CD71 and CD235a flow cytometry of primary erythroblasts at day 15 of differentiation. e, Representative Wright-Giemsa stains of primary erythroblasts at day 15 of differentiation. Experiments in c–e were performed on three donors/biological replicates with similar results. Scale bar, 25 μm.
