San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.
Vita-Salute San Raffaele University, Milan, Italy.
Nat Biotechnol. 2024 Jun;42(6):877-891. doi: 10.1038/s41587-023-01915-4. Epub 2023 Sep 7.
Base and prime editors (BEs and PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses and genotoxicity. Here, we compared state-of-the-art BEs and PEs and Cas9 in human hematopoietic stem and progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes and on-target and genome-wide genotoxicity. BEs and PEs induced detrimental transcriptional responses that reduced editing efficiency and hematopoietic repopulation in xenotransplants and also generated DNA double-strand breaks and genotoxic byproducts, including deletions and translocations, at a lower frequency than Cas9. These effects were strongest for cytidine BEs due to suboptimal inhibition of base excision repair and were mitigated by tailoring delivery timing and editor expression through optimized mRNA design. However, BEs altered the mutational landscape of hematopoietic stem and progenitor cells across the genome by increasing the load and relative proportions of nucleotide variants. These findings raise concerns about the genotoxicity of BEs and PEs and warrant further investigation in view of their clinical application.
碱基编辑和先导编辑（BEs 和 PEs）可能比基于核酸酶的方法提供更精确的基因工程，因为它们绕过了对 DNA 双链断裂的依赖。然而，关于它们的细胞反应和遗传毒性知之甚少。在这里，我们比较了最先进的 BEs 和 PEs 以及 Cas9 在人造血干细胞和祖细胞中的编辑效率、细胞毒性、转录组变化以及靶向和全基因组遗传毒性。BEs 和 PEs 诱导了有害的转录反应，降低了编辑效率和异种移植中的造血重建效率，并且以低于 Cas9 的频率产生 DNA 双链断裂和遗传毒性副产物，包括缺失和易位。由于碱基切除修复的抑制作用不理想，胞嘧啶 BEs 的这些作用最强，并通过优化 mRNA 设计来调整递送时间和编辑表达来减轻。然而，BEs 通过增加核苷酸变异的负荷和相对比例来改变造血干细胞和祖细胞的突变景观。这些发现引起了对 BEs 和 PEs 的遗传毒性的关注，并鉴于其临床应用需要进一步研究。
