Yang Fei, Wang Yiyun, Wang Qiudao, Pang Jingtao, Liu Guolong, Yang Yang, Qin Shenguang, Zhang Ying, Lai Yongrong, Fu Bin, Zhu Yating, Wang Mengyao, Kurita Ryo, Nakamura Yukio, Liang Dan, Wu Yuxuan
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
BRL Medicine Inc., Shanghai 201108, China.
Life Med. 2023 Nov 13;2(5):lnad042. doi: 10.1093/lifemedi/lnad042. eCollection 2023 Oct.
DNA double-strand breaks (DSBs) induced by gene-editing tools are primarily repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR) using synthetic DNA templates. However, error-prone NHEJ may result in unexpected indels at the targeted site. For most genetic disorders, precise HDR correction using exogenous homologous sequence is ideal. But, the therapeutic application of HDR might be especially challenging given the requirement for the codelivery of exogenous DNA templates with toxicity into cells, and the low efficiency of HDR could also limit its clinical application. In this study, we efficiently repair pathogenic mutations in coding regions of hematopoietic stem cells (HSCs) using CRISPR/Cas9-mediated gene conversion (CRISPR/GC) using the paralog gene as the internal template. After transplantation, these edited HSCs successfully repopulate the hematopoietic system and generate erythroid cells with significantly reduced thalassemia propensity. Moreover, a range of pathogenic gene mutations causing β-thalassemia in HBB coding regions were effectively converted to normal wild-type sequences without exogenous DNA templates using CRISPR/GC. This highlights the promising potential of CRISPR/GC, independent of synthetic DNA templates, for genetic disease gene therapy.
基因编辑工具诱导产生的DNA双链断裂（DSB）主要通过非同源末端连接（NHEJ）或利用合成DNA模板的同源定向修复（HDR）来修复。然而，容易出错的NHEJ可能会在靶向位点导致意外的插入或缺失。对于大多数遗传疾病而言，使用外源性同源序列进行精确的HDR校正最为理想。但是，鉴于需要将具有毒性的外源性DNA模板共导入细胞，HDR的治疗应用可能极具挑战性，而且HDR的低效率也可能限制其临床应用。在本研究中，我们利用旁系同源基因作为内部模板，通过CRISPR/Cas9介导的基因转换（CRISPR/GC）有效修复了造血干细胞（HSC）编码区的致病突变。移植后，这些经过编辑的HSC成功重建了造血系统，并生成了地中海贫血倾向显著降低的红细胞。此外，利用CRISPR/GC，无需外源性DNA模板，一系列在HBB编码区导致β地中海贫血的致病基因突变被有效转换为正常的野生型序列。这凸显了不依赖合成DNA模板的CRISPR/GC在遗传疾病基因治疗方面的巨大潜力。
