Targeted gene editing in hematopoietic stem cells (HSCs) is a promising treatment for several diseases. However, the limited efficiency of homology-directed repair (HDR) in HSCs and the unknown impact of the procedure on clonal composition and dynamics of transplantation have hampered clinical translation. Here, we apply a barcoding strategy to clonal tracking of edited cells (BAR-Seq) and show that editing activates p53, which substantially shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserve multilineage and self-renewing capacity. Transient p53 inhibition restored polyclonal graft composition. We increased HDR efficiency by forcing cell-cycle progression and upregulating components of the HDR machinery through transient expression of the adenovirus 5 E4orf6/7 protein, which recruits the cell-cycle controller E2F on its target genes. Combined E4orf6/7 expression and p53 inhibition resulted in HDR editing efficiencies of up to 50% in the long-term human graft, without perturbing repopulation and self-renewal of edited HSCs. This enhanced protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.

造血干细胞（HSC）中的靶向基因编辑是治疗多种疾病的有前途的治疗方法。但是，HSC中同源指导修复（HDR）的效率有限，以及该程序对克隆成分和移植动力学的未知影响，阻碍了临床翻译。在这里，我们将条形码策略应用于编辑细胞的克隆追踪（BAR-Seq），并显示编辑激活p53，这会大大缩小造血小鼠的HSC克隆库，尽管嫁接的编辑克隆保留了多系和自我更新的能力。瞬时p53抑制恢复了多克隆移植物的组成。我们通过瞬时表达腺病毒5 E4orf6 / 7蛋白来强迫细胞周期进程和上调HDR机器的组件，从而提高了HDR效率，它在其靶基因上募集细胞周期控制器E2F。结合的E4orf6 / 7表达和p53抑制作用在长期的人类移植物中导致HDR编辑效率高达50％，而不会干扰已编辑的HSC的繁殖和自我更新。这种增强的协议应扩大HSC基因编辑的适用性，并为临床翻译铺平道路。