Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation.

Autor: Wang J; Institut Jacques Monod, Université de Paris Cité, Paris, France.; Institut de Biologie, Ecole Normale Supérieure, Université PSL, Paris, France.; Department of Radiation Oncology, School of Medicine, Stanford University, Stanford, CA, USA.; School of Medicine, Stanford University, Stanford, CA, USA., Le Gall J; Institut de Biologie, Ecole Normale Supérieure, Université PSL, Paris, France., Frock RL; Programme « Equipe Labélisée » de la Ligue Nationale Contre le Cancer, Paris, France., Strick TR; Institut de Biologie, Ecole Normale Supérieure, Université PSL, Paris, France. strick@ens.psl.eu.; Programme « Equipe Labélisée » de la Ligue Nationale Contre le Cancer, Paris, France. strick@ens.psl.eu.
Jazyk: angličtina
Zdroj: Nature structural & molecular biology [Nat Struct Mol Biol] 2023 Nov; Vol. 30 (11), pp. 1707-1718. Date of Electronic Publication: 2023 Oct 12.
DOI: 10.1038/s41594-023-01104-6
Abstrakt: 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.
(© 2023. The Author(s).)
Databáze: MEDLINE
Externí odkaz: