DNA Supercoiling Drives CRISPR Off-Target Cuts, Study Finds
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- MRC Laboratory of Medical Sciences, Imperial College London, and University of Sheffield researchers published in Nature showing that DNA supercoiling—not just sequence—is a primary driver of CRISPR-Cas9 off-target editing errors.
- Quentin Smith and the team engineered nanometer-scale DNA minicircles small enough for cryo-EM visualization while still holding a supercoiled state, overcoming a 'Goldilocks' sizing problem that had blocked structural studies of Cas9 on twisted DNA.
- Professor David Rueda's group found that the same DNA sequence remains completely intact in linear form but gets cut by Cas9 once supercoiled, implicating DNA topology in unintended edits rather than sequence alone.
- Cryo-electron microscopy images revealed Cas9 restructures its geometry on supercoiled DNA, repositioning the HNH nuclease domain toward the cut site and tolerating new types of mismatches not seen in earlier linear-DNA structures.
- Off-target CRISPR errors cost the gene-editing industry an estimated $0.3 to $0.9 billion per year in profiling, guide redesign, and delays, according to Rueda.
- Most high-fidelity Cas9 variants were engineered using linear DNA structures, raising the prospect that they may not deliver the same off-target reductions inside cells where DNA is naturally supercoiled.
Why it matters: Off-target errors already cost the gene-editing industry an estimated $0.3–$0.9 billion per year, and the study shows the problem is baked into how current high-fidelity Cas9 variants were designed—against flat linear DNA rather than the twisted, buckled DNA inside real cells. That gives enzyme engineers a concrete structural target for redesigning CRISPR tools that actually perform as expected in patients.
