In the ever-evolving landscape of genome editing, CRISPR–Cas9 technology stands out as a groundbreaking tool, capable of precise and targeted DNA modification. However, despite its revolutionary potential, challenges remain—particularly the unintended cleavage of off-target DNA sequences. Recent research uncovers how DNA supercoiling influences Cas9’s off-target activity, marking a pivotal advance in understanding and potentially mitigating these unwanted effects.
CRISPR–Cas9 functions by forming R-loops, a structure where the guide RNA pairs with a complementary DNA strand, enabling site-specific cleavage. Traditionally, off-target activity was attributed to mismatches between the guide RNA and DNA target, but the molecular triggers behind these erroneous cuts were enigmatic. Breaking new ground, researchers employed programmable negatively supercoiled (−SC) DNA minicircles to mimic natural DNA topologies more faithfully than linear substrates, providing fresh perspectives on Cas9’s behavior within a physiologically relevant context.
High-resolution atomic force microscopy (AFM) revealed that supercoiling transforms relaxed circular DNA minicircles into collapsed or double-denatured conformations. Notably, Cas9 binding to these (−)SC minicircles prompted an open ‘diamond-ring’-shaped architecture—a direct consequence of local DNA topology relaxation characterized by a ∆Lk of −2, corresponding to complete R-loop formation. This structural adjustment underscores how DNA supercoiling modulates Cas9 engagement at a fundamental level.
Advancing from AFM observations, cryo-electron microscopy (cryo-EM) provided atomic details of the Cas9 complex bound to (−)SC DNA. While the Cas9 complex maintained a bilobed configuration reminiscent of its structure on linear DNA, significant conformational shifts emerged: the HNH nuclease domain moved approximately 15 Å closer to the scissile phosphate on the target strand, effectively positioning Cas9 in a catalytically primed state. This nuanced understanding clarifies how DNA topology affects enzyme activation dynamics previously unresolved in studies relying on linear substrate models.
Further structural insights highlighted the entire non-target strand (NTS) path traced through the PAM-interacting domain (PID), RuvC nuclease domain, and PAM-distal duplex, which had eluded detection in earlier Cas9-DNA structures. These observations suggest that supercoiled DNA creates a more ‘relaxed’ R-loop checkpoint, facilitating the accommodation of diverse mismatches, especially beyond the seed region—a critical segment close to the protospacer adjacent motif (PAM).
Corroborating these structural findings, single-molecule fluorescence resonance energy transfer (smFRET) studies revealed dynamic conformations and domain movements within Cas9 when complexed with supercoiled off-target sequences. The conformational plasticity of the HNH domain during off-target recognition aligns with allosteric activation models and experiments indicating accelerated cleavage rates on (−)SC DNA substrates compared to linear counterparts. Collectively, these data support a model where DNA supercoiling lowers the energetic barrier for R-loop formation, enhancing Cas9 off-target activity.
Intriguingly, two distinct (−)SC off-target Cas9 structures illuminated the mechanisms by which various mismatches are tolerated, including those in the seed region traditionally thought to be less permissive. These structures revealed unconventional, non-canonical base-pairing involving tautomeric Watson–Crick-like pairs and purine tautomer clashes, suggesting that negative supercoiling promotes flexible R-loop architectures accommodating steric hindrances that linear DNA cannot. Importantly, methods hint at a displacement of approximately 5.4 Å in the REC3 domain, potentially modulating the recognition and tolerance of PAM-distal mismatches.
The enhanced mobility of the HNH and REC2 domains in off-target complexes corresponds tightly with the observed allosteric activation paradigms. This domain flexibility, captured both in static structures and smFRET dynamics, may facilitate partial but catalytically competent R-loop formation, even when mismatches exist. Thus, negative supercoiling emerges as a critical factor driving the balance between specificity and efficiency in Cas9-mediated cleavage.
Post-cleavage analyses demonstrated that (−)SC substrates preserve the diamond-ring-like configuration post-DNA strand cleavage, and interestingly exhibit evidence of staggered cleavage events with a second possible cutting site. This structural retention supports biochemical and in vivo data indicating that Cas9 remains tightly bound to cleaved DNA and must be actively displaced for DNA replication or transcription to proceed, emphasizing supercoiling’s role in maintaining the structural integrity of Cas9-DNA complexes.
Beyond these structural variations, the study highlights a relaxed base-pairing requirement in the PAM-distal region under negatively supercoiled conditions, reducing the strictness of the classic ~15 bp guide-RNA complementarity rule previously thought necessary for cleavage activity. This finding suggests the critical role of DNA topology in modulating Cas9 specificity thresholds, with important ramifications for the design of truncated guide RNAs intended to enhance specificity in gene editing applications.
While the supercoiling densities used (σ ≈ −0.167 and −0.099) may differ from some physiological contexts, studies indicate that σ ≈ −0.06 is representative of genomic DNA superhelicity in vivo, meaning that the observed structural mechanisms likely approximate physiologically relevant states. These insights open new avenues for exploring how different chromatin states and DNA topologies influence genome-wide off-target events, thereby offering exciting prospects for tailoring CRISPR tools with improved fidelity.
In essence, these investigations provide a compelling structural and mechanistic framework for understanding how DNA supercoiling inherently shapes Cas9 specificity and allosteric activation. This knowledge not only advances the fundamental science of CRISPR function but also informs the next generation of high-fidelity Cas9 variants with reduced off-target risks, crucial for therapeutic genome editing. Moreover, the innovative use of (−)SC minicircles as model substrates provides a versatile platform to further dissect DNA topology influences across myriad DNA-binding proteins.
As CRISPR technologies edge closer to clinical translation, mechanistic revelations such as these will prove invaluable. By harnessing topological cues intrinsic to the genome, scientists can refine editing precision, minimizing collateral genomic disturbances while maximizing therapeutic efficacy. This study marks an important step towards safer gene-editing strategies and underscores the complex interplay between DNA structure and molecular machines navigating the genome.
Subject of Research:
Supercoiling-induced modulation of CRISPR–Cas9 off-target cleavage activity.
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
DOI:
https://doi.org/10.1038/s41586-026-10255-7
