ArticleProceedings of the National Academy of Sciences of the United States of America2020
Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 65 papers.
What it found
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
65 citing papers in PubMed.
- Gene editing of hematopoietic stem cells: applications and advances.International journal of hematology · 2026Review
- Stepwise DNA-unwinding gates TnpB genome-editing activity.Molecular cell · 2026Article
- The Single-Stranded DNA Platform: A Potential Broad-Spectrum Vector for Editing Genes in Prokaryotes: ssDNA May Break Through the Difficulties of Genetic Manipulation on Non-Model Bacteria.BioEssays : news and reviews in molecular, cellular and developmental biology · 2026Review
- Article
- Structural basis of AtCas9 recognition of PAM mutants in underwound DNA topology.Nature structural & molecular biology · 2026Article
- The interplay between supercoiling and DNA modifying enzymes at the single-molecule level.Scientific reports · 2026Article
- Article
- Cytoplasmic circular dsDNA is a key constituent of stress granules.bioRxiv : the preprint server for biology · 2026Article
- Stepwise DNA unwinding gates TnpB genome-editing activity.bioRxiv : the preprint server for biology · 2026Article
- CRISPR-based therapeutic genome editing for inherited blood disorders.Nature reviews. Drug discovery · 2025Review
- TOPO-seq reveals DNA topology-induced off-target activity by Cas9 and base editors.Nature chemical biology · 2025Article
- Off-target interactions in the CRISPR-Cas9 Machinery: mechanisms and outcomes.Biochemistry and biophysics reports · 2025Review
- Visualization of a multi-turnover Cas9 after product release.Nature communications · 2025Article
- A high-throughput single-molecule platform to study DNA supercoiling effect on protein-DNA interactions.Nucleic acids research · 2025Article
- Controlling DNA-RNA strand displacement kinetics with base distribution.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.Molecular cell · 2025Article
- Linking CRISPR-Cas9 double-strand break profiles to gene editing precision with BreakTag.Nature biotechnology · 2025Article
- Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner.Genome biology · 2025Article
- Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates.Nature communications · 2025Article
- DNA calorimetric force spectroscopy at single base pair resolution.Nature communications · 2025Article
5 more citing papers are in PubMed but not listed here.
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
6 authors.
Funding
Abstract
The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.
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