Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D Unit, AstraZeneca, Gothenburg, Sweden.
Department of Chemistry & Molecular Biology, University of Gothenburg, Gothenburg, Sweden.
Nat Commun. 2022 Mar 24;13(1):1240. doi: 10.1038/s41467-022-28771-1.
Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.
碱基编辑技术最近成为一种精确基因组编辑的新一代方法。在这里，我们利用 DNA 双链断裂 (DSB) 修复来开发两种策略，使用基于 SpCas9 核酸酶的碱基编辑器 (PEn) 进行精确的基因组插入。我们首先证明，通过同源依赖性 DSB 修复机制，PEn 与常规的碱基编辑指导 RNA (pegRNA) 结合可有效地促进短基因组插入。虽然 PEn 编辑会导致副产物水平增加，但它可以挽救与 Nickase 碱基编辑器配合使用性能不佳的 pegRNA。我们还提出了一种小分子方法，可提高 PEn 编辑的产物纯度。接下来，我们开发了一种不依赖同源性的 PEn 编辑策略，该策略通过非同源末端连接途径 (NHEJ) 在 DSB 处安装基因组插入物。最后，我们表明 PEn 介导的 DSB 插入可防止 Cas9 诱导的大片段缺失，并提供证据表明，Cas9 诱导的大片段缺失的机制之一是 Cas9 介导的连续切割。总之，这项工作通过利用包括 NHEJ 在内的不同 DNA 修复机制扩展了当前的碱基编辑工具包，NHEJ 是哺乳动物细胞中 DSB 修复的主要途径。
