Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere-distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function. [Display omitted] •Combined editing targeting the +58 and +55 enhancers augments HbF induction in HSPCs•Combined editing simultaneously disrupts critical TF binding sites at both enhancers•Gene editing without ex vivo culture minimizes micronuclei and long deletions•Gene editing without ex vivo culture preserves HSC repopulation and HbF potency Zeng et al. demonstrate that combined gene editing of BCL11A +58 and +55 enhancers disrupts two key TGN7–9WGATAR motifs and results in superior fetal hemoglobin (HbF) induction. Editing quiescent hematopoietic stem and progenitor cells (HSPCs) without ex vivo culture avoids unintended on-target genotoxicity, including micronuclei and long deletions.