ArticleProceedings of the National Academy of Sciences of the United States of America2017
Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 38 papers.
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Who cites it
38 citing papers in PubMed, 78 citations in OpenAlex.
- Exchange dynamics and kinetic control of gene regulation complexes.Nature reviews. Molecular cell biology · 2026Review
- CRISPR/Cas9 in perspective: evaluating efficacy, delivery methods, and ethical challenges in genome editing.Molecular biology reports · 2026Review
- CRISPR-Cas9 trans-cleavage is hindered by a flanked R-loop, an elongated spacer, and an inactive HNH domain.Nature communications · 2026Article
- Structural and functional bases of F. rodentium Cas9 provide insights into CRISPR-Cas protein engineering.Cell genomics · 2026Article
- CRISPR-Cas systems for plant virus management: detection, surveillance, and host resistance.Frontiers in plant science · 2026Review
- Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.Molecular cell · 2025Article
- CRISPR-Cas9-based electrochemical biosensor for the detection ofADMET & DMPK · 2025Article
- A long journey towards genome editing technologies in plants: a technical and critical review of genome editing technologies.Frontiers in genome editing · 2025Review
- The Many (Inter)faces of Anti-CRISPRs: Modulation of CRISPR-Cas Structure and Dynamics by Mechanistically Diverse Inhibitors.Biomolecules · 2023Review
- CRISPR/Cas9 therapeutics: progress and prospects.Signal transduction and targeted therapy · 2023Review
- G-quadruplex-guided RNA engineering to modulate CRISPR-based genomic regulation.Nucleic acids research · 2022Article
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- Programmable CRISPR-Cas9 microneedle patch for long-term capture and real-time monitoring of universal cell-free DNA.Nature communications · 2022Article
- CRISPR-Cas9 bends and twists DNA to read its sequence.Nature structural & molecular biology · 2022Article
- A tweak and a peek: How Cas9 pries open double-stranded DNA to check its sequence.Nature structural & molecular biology · 2022Article
- Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA.Nucleic acids research · 2022Article
- Computational Approaches for Designing Highly Specific and Efficient sgRNAs.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Efficient DNA interrogation of SpCas9 governed by its electrostatic interaction with DNA beyond the PAM and protospacer.Nucleic acids research · 2021Article
- Nonspecific interactions between SpCas9 and dsDNA sites located downstream of the PAM mediate facilitated diffusion to accelerate target search.Chemical science · 2021Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 2 countries.
Funding
Abstract
The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.
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Registered trials
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.