For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs in vivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2 months following transplantation, with little improvement via 53BP1 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs.

为了通过 CRISPR/同源定向修复 (HDR) 进行精确的基因组编辑，需要对长期移植造血干细胞 (LT-HSC) 进行有效且安全的编辑。尚未研究 HDR 对相关大型动物模型中真实 LT-HSC 克隆动态的影响。为了追踪 HDR 编辑细胞的输出和克隆性，并与体内慢病毒转导的 HSC 进行比较，我们开发了一种竞争性恒河猴 (RM) 自体移植模型，将转导有条形码的 GFP 慢病毒载体共输注 ( LV) 和 HDR 在 CD33 基因座进行编辑。与长期 LV 转导细胞的最小损失相比，CRISPR/HDR 编辑的细胞在移植后 2 个月显示出两个对数的减少，通过 53BP1 抑制几乎没有改善。与高度多克隆的 LV 转导的 HSC 相比，HDR 长期克隆性是寡克隆的。这些结果表明当前基因修饰方法对 HSC 的影响存在显着的临床相关差异。