ArticleNature structural & molecular biology2023
Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation.
Article in Nature structural & molecular biology, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 5 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
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Who cites it
5 citing papers in PubMed.
- Technical limitations of CRISPR-Cas9 genome editing in bacteria: challenges and future directions.Archives of microbiology · 2026Review
- 53BP1-RIF1 and DNA-PKcs show distinct genetic interactions with diverse chromosomal break repair outcomes.Nature communications · 2025Article
- Article
- Cleavage of DNA Substrate Containing Nucleotide Mismatch in the Complementary Region to sgRNA by Cas9 Endonuclease: Thermodynamic and Structural Features.International journal of molecular sciences · 2024Article
- DNA-PKcs suppresses illegitimate chromosome rearrangements.Nucleic acids research · 2024Article
Corrections and comments
- Erratum issued
Authors and funding
4 authors.
Funding
Abstract
Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by shifting (increasing or decreasing) the spacing between the RNA-DNA hybrid and the downstream canonical PAM. Up to 2-base 3' overhangs can be robustly generated via a 2-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR-SpCas9 reaction in real-time, structurally and kinetically monitoring and quantifying R-loop formation, the first and second DNA-incision events, and dissociation of the post-catalytic complex. Complex dissociation and release of broken DNA ends is a rate-limiting step of the reaction, and shifted SpCas9 is sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends.
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Registered trials
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