The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from β-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of β-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent β-thalassemia genotypes with currently available gene-editing technology.

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BCL11A, the major regulator of HbF(α 2 γ 2 ) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult-type HbA (α 2 β 2 ). To uncover how BCL11A initiates repression, we used CRISPR/Cas9, dCas9, dCas9-KRAB, and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established upon BCL11A depletion, and precedes γ-globin derepression and LCR-globin loop formation. Our findings reveal that the switch from fetal-to-adult globin gene expression within the >50 kb β-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.