伦敦帝国理工学院David S. Rueda团队的一项最新研究提出了超螺旋诱导CRISPR-Cas9脱靶活性的结构基础。相关论文于2026年3月25日发表在《自然》杂志上。

在这里，研究人员使用（−）SC DNA微环观察到DNA中的超螺旋驱动结构缺陷，这些缺陷通过Cas9结合得到解决。冷冻电子显微镜下结合在靶上和非靶构型的Cas9结构突出表明Cas9 HNH结构域处于更具催化能力的构象中。新的DNA-RNA错配几何结构被适应于整个原间隔物，而原间隔物邻近基序远端区域的结构可塑性依赖于拓扑结构。总之，他们的研究揭示了（−）SC诱导Cas9靶向的分子基础，并为设计具有拓扑背景的下一代高保真CRISPR效应物提供了框架。

据介绍，CRISPR-Cas9是一种强大的基因组编辑工具，但全基因组的脱靶活性可能会阻碍其治疗应用。负超螺旋（（−）SC）与脱靶活性有关，但缺乏分子水平的理解。

附：英文原文

Title: Structural basis of supercoiling-induced CRISPR–Cas9 off-target activity

Author: Smith, Quentin M., Whittle, Sylvia, Aramayo, Ricardo J., Rollins, Daniel E., Jalal, Adam S. B., Egharevba, Deborah I., Morris, Kyle L., Pyne, Alice L. B., Rueda, David S.

Issue&Volume: 2026-03-25

Abstract: CRISPR–Cas9 is a powerful genome-editing tool1, but genome-wide off-target activity can hinder therapeutic applications. Negative supercoiling (()SC) has been implicated in off-target activity, but a molecular-level understanding is lacking. Here, using ()SC DNA minicircles, we observe supercoiling-driven structural defects in the DNA that are resolved by Cas9 binding. Cryo-electron microscopy structures of Cas9 bound in both the on-target and off-target configurations highlight that the Cas9 HNH domain is poised in a more catalytically competent conformation. New DNA–RNA mismatch geometries are accommodated across the protospacer and structural plasticity in the protospacer adjacent motif distal region of the protospacer is topology dependent. Together, our study reveals the molecular basis for ()SC-induced Cas9 targeting and provides a framework for the design of next-generation high-fidelity CRISPR effectors with topological context.

DOI: 10.1038/s41586-026-10255-7

Source: https://www.nature.com/articles/s41586-026-10255-7