ArticleProceedings of the National Academy of Sciences of the United States of America2014
Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 263 papers, 1 of them a synthesis that pooled it.
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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.
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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
263 citing papers in PubMed, 1 synthesis or guideline pooled it.
- An overview and metanalysis of machine and deep learning-based CRISPR gRNA design tools.RNA biology · 2020Pooled it
- In-vivo CRISPR/Base-Editing "One-Shot" Therapies Targeting PCSK9.Current atherosclerosis reports · 2026Review
- A dynamically frustrated allosteric checkpoint consistent with conformational proofreading in CRISPR-Cas9.Nucleic acids research · 2026Article
- Size and sequence variation of the Lid of the Cas12a nuclease domain results in a nickase phenotype.Nucleic acids research · 2026Article
- Stepwise DNA-unwinding gates TnpB genome-editing activity.Molecular cell · 2026Article
- Helicity of the bridge helix of Cas12a regulates on-target DNA cleavage efficiency and off-target cleavage propensity.Biochemical and biophysical research communications · 2026Article
- Incorporating AI-optimized zinc finger proteins enhances the efficiencies and targeting ranges of miniature base editors.Nature communications · 2026Article
- Structural basis of AtCas9 recognition of PAM mutants in underwound DNA topology.Nature structural & molecular biology · 2026Article
- Article
- Operating CRISPR/Cas12a in a complex nucleic acid sequence background.Nucleic acids research · 2026Article
- CRISPR Interference to Inhibit Oncogenes for Cancer Therapy.International journal of molecular sciences · 2026Review
- Stepwise DNA unwinding gates TnpB genome-editing activity.bioRxiv : the preprint server for biology · 2026Article
- Conformational changes induced by K949A mutation in the CRISPR-Cas12a complex drives an effective target-binding mechanism.Current research in structural biology · 2025Article
- TOPO-seq reveals DNA topology-induced off-target activity by Cas9 and base editors.Nature chemical biology · 2025Article
- Rapid CRISPR-Cas9 target-strand nicking can provide phage resistance by reducing DNA abundance.Nucleic acids research · 2025Article
- Revolution and advances in gene editing and genomics technology for developing climate-resilient legume crops: developments and prospects.Plant molecular biology · 2025Review
- Structural and mechanistic insights into the sequential dsDNA cleavage by SpCas12f1.Nucleic acids research · 2025Article
- Design of function-regulating RNA via deep learning and AlphaFold 3.Briefings in bioinformatics · 2025Article
- 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
203 more citing papers are in PubMed but not listed here.
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Authors and funding
8 authors.
Funding
Abstract
Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems protect bacteria and archaea from infection by viruses and plasmids. Central to this defense is a ribonucleoprotein complex that produces RNA-guided cleavage of foreign nucleic acids. In DNA-targeting CRISPR-Cas systems, the RNA component of the complex encodes target recognition by forming a site-specific hybrid (R-loop) with its complement (protospacer) on an invading DNA while displacing the noncomplementary strand. Subsequently, the R-loop structure triggers DNA degradation. Although these reactions have been reconstituted, the exact mechanism of R-loop formation has not been fully resolved. Here, we use single-molecule DNA supercoiling to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade- and Cas9-based CRISPR-Cas systems. We find that the protospacer adjacent motif (PAM) affects primarily the R-loop association rates, whereas protospacer elements distal to the PAM affect primarily R-loop stability. Furthermore, Cascade has higher torque stability than Cas9 by using a conformational locking step. Our data provide direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Moreover, we introduce DNA supercoiling as a quantitative tool to explore the sequence requirements and promiscuities of orthogonal CRISPR-Cas systems in rapidly emerging gene-targeting applications.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.