title: Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations.
abstract: The 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 Mg2+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 Mg2+bound. The simulation results showed that binding of two Mg2+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.
authors.affiliation: Department of Pharmaceutical Sciences, University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Zuo
authors.affiliation: Department of Pharmaceutical Sciences, University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Liu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D020134:Catalytic Domain; D002414:Cations, Monovalent; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D053837:DNA Cleavage; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics; D008274:Magnesium / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D002414:Cations, Monovalent; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D008274:Magnesium