Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

• DOI: 10.1038/srep37584
• Publication Date: 2016-11-22T10:20:25Z
• Authors: Zhicheng Zuo; Jin Liu
• Keywords: Cleavage (geology); Guide RNA; Nuclease

Abstract The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as 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 catalytic Mg 2+ ions induce conformation changes toward active state. It also remains controversial whether generates blunt-ended or staggered-ended breaks overhangs DNA. To investigate these issues, here we performed first all-atom molecular dynamics simulations of spCas9-sgRNA-dsDNA system without bound. simulation results showed that binding two at RuvC domain site could lead to structurally energetically favorable coordination ready non-target strand cleavage. Importantly, demonstrated our Cas9-catalyzed cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.