Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Immune Regulation and Cancer, Berlin, Germany.
Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Genome Engineering & Disease Models, Berlin, Germany.
Sci Adv. 2022 Jun 3;8(22):eabm9106. doi: 10.1126/sciadv.abm9106.
While CRISPR-Cas9 is key for the development of gene therapy, its potential off-target mutations are still a major concern. Here, we establish a "spacer-nick" gene correction approach that combines the Cas9 nickase with a pair of PAM-out sgRNAs at a distance of 200 to 350 bp. In combination with adeno-associated virus (AAV) serotype 6 template delivery, our approach led to efficient HDR in human hematopoietic stem and progenitor cells (HSPCs including long-term HSCs) and T cells, with minimal NHEJ-mediated on-target mutations. Using spacer-nick, we developed an approach to repair disease-causing mutations occurring in the , , , and genes. We achieved gene correction efficiencies of 20 to 50% with minimal NHEJ-mediated on-target mutations. On the basis of in-depth off-target assessment, frequent unintended genetic alterations induced by classical CRISPR-Cas9 were significantly reduced or absent in the HSPCs treated with spacer-nick. Thus, the spacer-nick gene correction approach provides improved safety and suitability for gene therapy.
虽然 CRISPR-Cas9 是基因治疗发展的关键，但它潜在的脱靶突变仍然是一个主要关注点。在这里，我们建立了一种“间隔子切口”基因校正方法，该方法将 Cas9 切口酶与一对距离为 200 至 350bp 的 PAM-out sgRNA 结合使用。与腺相关病毒 (AAV) 血清型 6 模板递送相结合，我们的方法导致人造血干细胞和祖细胞 (HSPCs，包括长期 HSCs) 和 T 细胞中 HDR 的效率很高，而 NHEJ 介导的靶基因突变很少。利用间隔子切口，我们开发了一种方法来修复发生在 、 、 、 和 基因中的致病突变。我们实现了 20%至 50%的基因校正效率，同时最小化了 NHEJ 介导的靶基因突变。基于深入的脱靶评估，在使用间隔子切口处理的 HSPCs 中，经典 CRISPR-Cas9 诱导的频繁非预期遗传改变显著减少或不存在。因此，间隔子切口基因校正方法为基因治疗提供了更高的安全性和适用性。
