NatureMar 26, 2026

How DNA twisting causes CRISPR-Cas9 to cut unintended sites

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jun 27, 2026

Abstract

Essence

Negative DNA supercoiling appears to make CRISPR-Cas9 more able to accommodate off-target binding geometries.

Evidence

A structural cryo-electron microscopy and DNA minicircle study examined Cas9 bound to negatively supercoiled DNA in on-target and off-target configurations.

Caveat

The work defines a molecular mechanism in engineered DNA systems, not genome-wide editing outcomes in therapeutic cells or organisms.

Simplified

CRISPR-Cas9 is a powerful genome-editing tool, 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. 1

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How DNA twisting causes CRISPR-Cas9 to cut unintended sites

Abstract

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