In vivo selection for corrected ß-globin alleles after CRISPR/Cas9 editing in human sickle hematopoietic stem cells

Sickle Cell Disease (SCD), one of the world's most common genetic disorders, causes anemia and progressive multiorgan damage that typically shortens lifespan by decades; currently there is no broadly applicable curative therapy. Cas9 RNP-mediated gene editing with an ssDNA oligonucleotide donor yields more than 20% correction of the sickle mutation in long-term engrafting human HSCs. Using RNA-seq, we further find that in vivo erythroid differentiation markedly enriches for cells carrying corrected ß-globin alleles. Adoption of a high-fidelity Cas9 variant demonstrates that this approach can yield efficient editing with almost no off-target events. These findings indicate that the sickle mutation can be corrected in human HSCs at levels that are likely to be curative if translated into a therapy.

镰状细胞病（Sickle Cell Disease, SCD）是全球最常见的遗传性疾病之一，可引发贫血及进行性多器官损伤，通常会使患者寿命缩短数十年，目前尚无广泛适用的治愈性疗法。研究团队采用单链DNA寡核苷酸供体（ssDNA oligonucleotide donor）配合Cas9核糖核蛋白复合物（Cas9 RNP）介导的基因编辑技术，可使长期植入的人造血干细胞（hematopoietic stem cells, HSCs）中镰状突变的校正率超过20%。通过RNA测序（RNA-seq）进一步分析发现，体内红系分化过程会显著富集携带校正后β-珠蛋白等位基因的细胞。使用高保真Cas9变体的实验表明，该策略可实现高效基因编辑，且几乎无脱靶事件发生。上述研究结果证实，可在人造血干细胞中实现镰状突变的校正，若该技术转化为治疗手段，其校正水平有望达到治愈疾病的标准。