Highly efficient therapeutic gene editing of human hematopoietic stem cells

Yuxuan Wu, Jing Zeng, Benjamin P. Roscoe, Pengpeng Liu, Qiuming Yao, Cicera R. Lazzarotto, Kendell Clement, Mitchel A. Cole, Kevin Luk, Cristina Baricordi, Anne H. Shen, Chunyan Ren, Erica B. Esrick, John P. Manis, David M. Dorfman, David A. Williams, Alessandra Biffi, Carlo Brugnara, Luca Biasco, Christian BrendelLuca Pinello, Shengdar Q. Tsai, Scot A. Wolfe, Daniel E. Bauer

Research output: Contribution to journalArticlepeer-review

319 Scopus citations


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 cells2–6. 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.

Original languageEnglish
Pages (from-to)776-783
Number of pages8
JournalNature Medicine
Issue number5
StatePublished - 1 May 2019
Externally publishedYes


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