Attribution 4.0 International (CC BY 4.0)2022-09-302022-09-302016-11-22Zuo, Z., & Liu, J. (2016). Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations. Scientific reports, 5, 37584. https://doi.org/10.1038/srep375842045-2322https://hdl.handle.net/20.500.12503/31822The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg(2+) ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg(2+) bound. The simulation results showed that binding of two Mg(2+) ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.http://creativecommons.org/licenses/by/4.0/Bacterial Proteins / chemistryBacterial Proteins / geneticsCRISPR-Associated Protein 9Catalytic DomainCations, MonovalentDNA / chemistryDNA / geneticsDNA CleavageEndonucleases / chemistryEndonucleases / geneticsMagnesium / chemistryMolecular Dynamics SimulationRNA, Guide / chemistryArticle© The Author(s) 20165