Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2,3,4,5,6. CRISPR–Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data that support the findings of this study are available within the paper and its supplementary information files. The deep sequencing data that support the findings of this study are publicly accessible from the National Center for Biotechnology Information Bioproject database with the accession number PRJNA517275, including the editing efficiency, pre- or post-mice-transplant data in Figs. 1–4 and the off-target assessment in Extended Data Fig. 6. The analytical results and statistics used to generate Figs. 1–4 and Extended Data Fig. 6 are provided in Supplementary Table 9. There are no restrictions on availability of the data from this study.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We thank D. Chui for genetic analyses, N. Barteneva for imaging flow cytometry analysis, R. Mathieu for flow cytometry assistance, Z. Herbert for deep sequencing, and G. Menard and R. Rosales for help with hemoglobin HPLC. We appreciate useful discussions with J. Hsu, B. Croker, C. Lindsley, K. Holden, M. Hoban, M. Canver, and S. Orkin. This project was funded in part by the Translational Research Program at BCH. D.A.W. and C. Brendel were supported by NHLBI (grant no. U01HL11772) and D.A.W. and E.B.E. by NHLBI (grant no. R01HL137848). The trial for SCD HSPC procurement was supported by research funding from bluebird bio to A.B. L.P. was supported by NHGRI (grant no. R00HG008399). S.A.W. was supported by NIAID (grant no. R01AI117839) and NIGMS (grant no. R01GM115911). D.E.B. was supported by NIDDK (grant nos. K08DK093705 and R03DK109232), NHLBI (grant nos. DP2OD022716, P01HL053749 and P01HL032262), Harvard Stem Cell Institute Seed Grant, St. Jude Children’s Research Hospital Collaborative Research Consortium, Burroughs Wellcome Fund, American Society of Hematology, and the Doris Duke Charitable, Charles H. Hood, and Cooley’s Anemia Foundations.