In a groundbreaking study published recently in Nature, researchers have unveiled the intricate molecular mechanisms behind CRISPR–Cas9 off-target activity, emphasizing the crucial role of DNA topology in guiding cleavage precision. CRISPR–Cas9 has revolutionized genome editing due to its remarkable targeting versatility. However, unintended off-target cleavages have posed significant challenges for therapeutic safety. This new work illuminates how negatively supercoiled DNA substrates promote off-target cleavage, reshaping our understanding of Cas9’s behavior inside cells and providing pathways for next-generation genome editing tools.
The team approached this complex question by engineering small, negatively supercoiled (−)SC DNA minicircles that serve as biologically relevant Cas9 substrates. Unlike traditional linear DNA constructs used in many previous studies, these minicircles mimic the torsional strain experienced by genomic DNA in vivo. High-resolution atomic force microscopy (AFM) revealed that supercoiling transforms relaxed DNA circles into a collapsed configuration, known as a double-denatured state, confirming molecular dynamics simulations. This topology distinctly changes how Cas9 interacts with DNA, promoting an open diamond-ring-like configuration that is directly linked to local topological relaxation and R-loop stabilization.
To capture the structural details underlying this unique interaction, the researchers employed cryogenic electron microscopy (cryo-EM) on catalytically inactive Cas9 (dCas9) bound to these (−)SC minicircles. The resulting density maps confirmed the diamond-ring-like arrangement and showed that, globally, Cas9 maintains its bilobed architecture seen on linear DNA. However, a critical difference emerged: the HNH nuclease domain swings approximately 15 angstroms closer to the target strand’s scissile phosphate group, positioning the enzyme closer to an activated cleavage state. This proximity likely lowers the activation energy necessary for cutting, providing a structural explanation for previously observed acceleration of cleavage in supercoiled contexts.
Earlier structures of Cas9 bound to linear or partially duplexed DNA substrates showed a more flexible and poorly resolved HNH domain, suggesting incomplete activation states. This new (−)SC-bound structure contrasts sharply, revealing better-defined conformations of the non-target strand’s path through Cas9, including the PAM-interacting domain (PID), the RuvC nuclease domain, and the distal double-stranded DNA duplex. These orders-out-of-chaos observations suggest that DNA supercoiling fosters a more precise and stable Cas9-DNA complex architecture, shedding light on its mechanistic basis for off-target tolerance.
Furthermore, the study highlights dynamic domain interplay within Cas9 upon binding (−)SC DNA. Molecular dynamics simulations and single-molecule fluorescence resonance energy transfer (smFRET) experiments showed conserved allosteric activation pathways between the REC2 and HNH domains. Supercoiling appears to lower the kinetic barriers for R-loop formation, facilitating faster cleavage while maintaining fidelity mechanisms. Interestingly, off-target sequences exhibit increased HNH and REC2 domain mobility, suggesting a complex conformational energy landscape where Cas9 dynamically balances cleavage efficacy with mismatch tolerance.
Delving deeper, the authors solved two high-resolution structures of Cas9 bound to (−)SC off-target substrates with diverse mismatches across the protospacer, including the crucial PAM-distal seed region. They observed unconventional base-pairing geometries such as tautomeric Watson–Crick-like base pairs and purine tautomer clashes within the R-loop, uncovering a surprising plasticity that accommodates mismatches without fully sacrificing enzymatic activity. This contrasts markedly with prior studies using linear DNA, where PAM-distal mismatches are typically rigidly resolved via RuvC domain rearrangements, underscoring the role of DNA topology in modulating Cas9 specificity.
Another intriguing finding was the displacement of the REC3 domain by roughly 5.4 Å in off-target complexes, implicating this domain in modulating mismatch tolerance. The more flexible DNA topology likely allows Cas9 to maintain stable R-loop structures despite imperfect base pairing, enhancing off-target cleavage probability. These insights pave the way for engineering Cas9 variants with improved specificity by targeting domain dynamics and DNA topology recognition.
The authors also investigated post-cleavage conformations of Cas9 on (−)SC DNA, revealing that the enzyme-product complex preserves the diamond-ring structure rather than dissociating immediately. This behavior aligns with previous single-molecule tweezing and in vivo studies indicating Cas9 remains persistently bound after cleavage and must be actively removed by cellular replication or transcription machinery. Notably, they detected alternative potential cleavage sites consistent with a staggered cut mechanism, expanding the mechanistic understanding of Cas9-mediated DNA scission.
From a therapeutic perspective, these discoveries emphasize the importance of DNA topology in modulating CRISPR-Cas9 activity within chromatin contexts. While cellular DNA supercoiling densities vary, the supercoiling levels studied (σ = −0.099 to −0.167) approach physiological conditions, validating the biological relevance of the findings. Future studies expanding genome-wide off-target profiling under varying supercoiling states may unlock new specificity paradigms during gene editing.
Altogether, this work establishes a comprehensive mechanistic framework integrating DNA topology, Cas9 conformational dynamics, and mismatch tolerance. The findings reveal how negatively supercoiled DNA lowers energetic barriers and reshapes the Cas9 energy landscape, promoting off-target activity through non-canonical base pairing within the R-loop and allosteric domain rearrangements. Such insights hold significant promise for rationally designing next-generation high-fidelity CRISPR effectors with minimized off-target effects.
In conclusion, the marriage of biophysical techniques including AFM, cryo-EM, MD simulations, and smFRET has provided a detailed structural and dynamic picture of Cas9 function modulated by DNA supercoiling. By targeting DNA topology as a regulatory axis, future genome editing strategies can harness finer control over Cas9 specificity and activity. These programmable (−)SC minicircles also constitute a powerful platform for broader studies probing the impact of DNA topology on various DNA-binding proteins and enzymatic systems.
This seminal study thus marks a major leap forward in genome editing research, paving the way for safer and more precise therapeutic applications of CRISPR technology. The structural basis of topology-induced off-target cleavage elucidated here rewrites our understanding of Cas9’s molecular choreography in the cell, catalyzing the next frontier of gene editing innovation.
Subject of Research: Structural mechanisms underpinning supercoiling-induced off-target activity of CRISPR–Cas9.
Article Title: Structural basis of supercoiling-induced CRISPR–Cas9 off-target activity.
Article References:
Smith, Q.M., Whittle, S., Aramayo, R.J. et al. Structural basis of supercoiling-induced CRISPR–Cas9 off-target activity. Nature (2026). https://doi.org/10.1038/s41586-026-10255-7
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