日本东京大学Osamu Nureki等研究人员合作揭示先导编辑器介导pegRNA引导的反转录的结构基础。2024年5月29日，《自然》杂志在线发表了这项成果。

研究人员表示，由化脓性链球菌Cas9缺口酶（nSpCas9）和工程化的莫罗尼小鼠白血病病毒反转录酶（M-MLV RT）组成的，先导编辑器系统与先导编辑向导RNA（pegRNA）合作，促进了活细胞中各种精确的基因组编辑。然而，由于缺乏结构信息，人们对先导编辑引导的 pegRNA 反转录的分子机制仍然知之甚少。

研究人员展示了 SpCas9-M-MLV RTΔRNaseH-pegRNA-靶DNA 复合物在多种状态下的冷冻电镜结构。终止结构以及功能分析显示，M-MLV RT 将反转录扩展到了预期的位点之外，导致支架衍生的结合，从而在靶基因座上造成了不希望的编辑。此外，启动前、启动和延伸状态的结构比较表明，在反转录过程中，M-MLV RT 相对于 SpCas9 保持一致的位置，而 pegRNA 合成的 DNA 杂复合物则沿着 SpCas9 的表面堆积。

根据这些结构见解，研究人员合理地设计了 pegRNA 变体和先导编辑器变体，其中 M-MLV RT 与 SpCas9 融合。总之，这些发现从结构上揭示了先导编辑的逐步机制，并将为开发多功能先导编辑工具箱铺平道路。

附：英文原文

Title: Structural basis for pegRNA-guided reverse transcription by a prime editor

Author: Shuto, Yutaro, Nakagawa, Ryoya, Zhu, Shiyou, Hoki, Mizuki, Omura, Satoshi N., Hirano, Hisato, Itoh, Yuzuru, Zhang, Feng, Nureki, Osamu

Issue&Volume: 2024-05-29

Abstract: The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells1. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9–M-MLV RTΔRNaseH–pegRNA–target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA–synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.

DOI: 10.1038/s41586-024-07497-8

Source: https://www.nature.com/articles/s41586-024-07497-8