Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) serve as adaptive bacterial immune systems against viral infections and have been repurposed as powerful RNA-guided genome-editing tools for eukaryotic cells. Precise target recognition relies on Streptococcus pyogenes Cas9 (SpCas9, an RNA-guided endonuclease) recognizing a specific protospacer adjacent motif (PAM, a short 5′-NGG-3′ DNA sequence required for target binding) and unwinding the adjacent 20-base-pair target sequence to form an R-loop (a three-stranded nucleic acid structure consisting of a DNA-RNA hybrid and a displaced single DNA strand). Cleavage of double-stranded DNA is executed by two distinct endonucleolytic domains: HNH (the nuclease domain responsible for cutting the target DNA strand) and RuvC (the nuclease domain responsible for cutting the non-target DNA strand). However, imperfect sequence complementarity between the guide RNA and target DNA or alternative PAM recognition can trigger off-target cleavage (unintended DNA cleavage at non-targeted genomic sites), representing a major limitation for precise genome engineering.
Although DNA unwinding driven by mechanical or biochemical forces enhances off-target cleavage across genome-wide targets, the precise molecular mechanism by which negative supercoiling ((-)SC), modulates Cas9 binding, allosteric activation (structural communication between distant protein regions to regulate catalytic activity), and cleavage has remained unresolved due to a lack of structural data on negatively supercoiled substrates. In living cells, supercoiling density (the degree of torsional strain in a DNA molecule) is regulated by topoisomerases (enzymes that alter the topological state of DNA) and is fundamentally involved in physiological processes, including gene expression, DNA replication, repair, and chromatin organization. To overcome the absence of topological context in structural biology, researchers aimed to elucidate how negative supercoiling affects target recognition, structural reorganization, mismatch tolerance, and catalytic activation of Cas9.
To investigate these topological mechanisms, researchers engineered 126-base-pair negatively supercoiled DNA minicircles (small, topologically constrained circular DNA molecules) carrying on-target and off-target sequences, including the well-characterized EMX1-1 target. Structural alterations in relaxed and supercoiled minicircles were evaluated using high-resolution atomic force microscopy (AFM, a scanning probe microscopy technique used to image nanoscale biomolecular structures), and high-resolution structures of dead Cas9 (dCas9, a catalytically inactive Cas9 variant) and wild-type Cas9 bound to supercoiled minicircles were determined using cryo-electron microscopy (cryo-EM, an imaging technique used to determine three-dimensional structures of macromolecular complexes at near-atomic resolution). Furthermore, the researchers utilized single-molecule fluorescence resonance energy transfer (smFRET, a technique measuring energy transfer between fluorophores to monitor intramolecular distance changes) integrated with optical tweezers (a single-molecule tool using laser beams to physically trap and manipulate microscopic objects) to capture real-time conformational dynamics of the HNH domain on negatively supercoiled λ-DNA under controlled torsional strain.
The cryo-EM structures revealed that binding of dCas9 to on-target negatively supercoiled minicircles resolves supercoiling-induced structural defects, restoring an open circular geometry while positioning the HNH domain approximately 15 angstroms closer to the target strand scissile phosphate (the specific phosphodiester bond targeted for cleavage).” target strand scissile phosphate (the specific phosphodiester bond targeted for cleavage). In off-target complexes containing multiple seed-region mismatches (proximal seed region ), negative supercoiling facilitated R-loop formation by accommodating non-canonical base-pair geometries, such as a guanine-guanine purine clash at position 3, with the protospacer DNA guanine adopting a syn-conformation (a rotational orientation of a nucleobase relative to its sugar ring) to form a Hoogsteen base pair with the guide RNA guanine, alongside protonated and tautomeric wobble base pairs. Furthermore, topological stress increased structural flexibility at the PAM-distal region (the end of the target sequence furthest from the PAM), allowing Cas9 to cleave negatively supercoiled minicircles with guide RNAs of 16 nucleotides and to nick them with a 14-nucleotide guide. Single-molecule FRET and optical tweezer experiments revealed revealed dynamic conformational changes of the HNH domain under negative supercoiling, consistent with allosteric activation. Cleavage experiments further showed that off-target cleavage rates under negative supercoiling were generally approximately 100-fold compared to linear off-target DNA.
These findings provide astructural and mechanical framework governing supercoiling-induced off-target activity in CRISPR-Cas9 systems. Notably, on-target negatively supercoiled minicircles were linearized in less than 20 seconds, with an observed rate constant of kobs ≥ 2.5 ± 0.6 min−1, approximately 10-fold faster than the corresponding linear substrate. By demonstrating how negative DNA supercoiling promotes R-loop formation and can facilitate Cas9 activation even in the presence of extensive seed and distal mismatches, the study highlights importance of considering DNA topology into target profiling. Ultimately, these structural insights and programmable supercoiled DNA minicircle platforms provide foundational data for evaluating DNA-binding proteins and designing high-fidelity CRISPR effectors tailored to physiological topological contexts.
Author: Elinsu Ak
Editor: Nur Tanem Altundaş
Reference: Smith, Q. M., Whittle, S., Aramayo, R. J., Rollins, D. E., Jalal, A. S. B., Egharevba, D. I., Morris, K. L., Pyne, A. L. B., & Rueda, D. S. (2026). Structural basis of supercoiling-induced CRISPR-Cas9 off-target activity. Nature, 653, 627–635. https://doi.org/10.1038/s41586-026-10255-7
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