ArticleThe EMBO journal2019
CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion.
Article in The EMBO journal, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 59 papers.
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Who cites it
59 citing papers in PubMed.
- Automated kinetic-scheme-free sorting of single-molecule fluorescence events using a deep learning based hidden-state method.Nature communications · 2026Article
- Intersegmental transfers drive target search in an RNA-targeting CRISPR system.bioRxiv : the preprint server for biology · 2026Article
- CRISPR diagnostics: from trans-nuclease activity to cancer diagnosis.Cell & bioscience · 2026Review
- Operating CRISPR/Cas12a in a complex nucleic acid sequence background.Nucleic acids research · 2026Article
- Better data for better predictions: data curation improves deep learning for sgRNA/Cas9 prediction.PeerJ · 2026Article
- GAGA zinc finger transcription factor searches chromatin by 1D-3D facilitated diffusion.Nature structural & molecular biology · 2025Article
- ADARs: pleiotropy in function, versatility in application.Nucleic acids research · 2025Review
- Mechanism of Cas9 inhibition by AcrIIA11.Nucleic acids research · 2025Article
- Article
- CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.Nucleic acids research · 2025Article
- Simulations predict stronger CRISPRi transcriptional repression in plants for identical than heterogeneous gRNA target sites.Synthetic biology (Oxford, England) · 2025Article
- Single-molecule perspectives of CRISPR/Cas systems: target search, recognition, and cleavage.BMB reports · 2025Review
- Deep learning based local feature classification to automatically identify single molecule fluorescence events.Communications biology · 2024Article
- Conformational dynamics of CasX (Cas12e) in mediating DNA cleavage revealed by single-molecule FRET.Nucleic acids research · 2024Article
- Harnessing the evolving CRISPR/Cas9 for precision oncology.Journal of translational medicine · 2024Review
- CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.bioRxiv : the preprint server for biology · 2024Article
- Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a.Nucleic acids research · 2024Article
- Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.Nature communications · 2024Article
- RNA compaction and iterative scanning for small RNA targets by the Hfq chaperone.Nature communications · 2024Article
- Engineering Cas9: next generation of genomic editors.Applied microbiology and biotechnology · 2024Review
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5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) nuclease has been widely applied in genetic engineering. Despite its importance in genome editing, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. In particular, because of the lack of a method with high spatio-temporal resolution, transient interactions between Cas9 and DNA could not be reliably investigated. It therefore remains controversial how Cas9 searches for protospacer adjacent motif (PAM) sequences. We have developed single-molecule Förster resonance energy transfer (smFRET) assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the PAM sequence weakly, yet probing neighboring sequences via facilitated diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with three-dimensional diffusion and thus is involved in PAM finding and consequently on-target binding. Our results imply that the neighboring sequences can be very important when choosing a target in genetic engineering applications.
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