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

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.

Metadata

Item Type:	Article
Authors/Creators:	Smith, Q.M. https://orcid.org/0000-0003-2967-9240 Whittle, S. https://orcid.org/0009-0008-9164-9513 Aramayo, R.J. Rollins, D.E. Jalal, A.S.B. Egharevba, D.I. https://orcid.org/0009-0007-5650-7790 Morris, K.L. https://orcid.org/0000-0002-1717-8134 Pyne, A.L.B. https://orcid.org/0000-0002-2658-8987 Rueda, D.S. https://orcid.org/0000-0003-4657-6323
Copyright, Publisher and Additional Information:	© The Author(s) 2026. Open Access: This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Keywords:	Biological physics; Single-molecule biophysics; Structural biology
Dates:	Submitted: 30 October 2024 Accepted: 9 February 2026 Published (online): 25 March 2026 Published: 14 May 2026
Institution:	The University of Sheffield
Academic Units:	The University of Sheffield > Faculty of Engineering (Sheffield) > School of Chemical, Materials and Biological Engineering The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Materials Science and Engineering (Sheffield)
Funding Information:	Funder Grant number Engineering and Physical Sciences Research Council EP/P02470X/1 Engineering and Physical Sciences Research Council EP/R00661X/1 UK RESEARCH AND INNOVATION MR/W00738X/1
Date Deposited:	09 Apr 2026 15:14
Last Modified:	15 May 2026 11:39
Status:	Published
Publisher:	Springer Science and Business Media LLC
Refereed:	Yes
Identification Number:	10.1038/s41586-026-10255-7
Related URLs:	PubMed URL Dataset Dataset Dataset Dataset Dataset Dataset Dataset Dataset Dataset Software or Code
Open Archives Initiative ID (OAI ID):	oai:eprints.whiterose.ac.uk:239875

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

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

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