Open AccessCas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations
Author(s) -
Zhicheng Zuo,
Jin Liu
Publication year - 2016
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/srep37584
Subject(s) - cas9 , cleavage (geology) , guide rna , genome editing , dna , endonuclease , crispr , chemistry , restriction enzyme , nuclease , biophysics , computational biology , microbiology and biotechnology , biology , gene , biochemistry , paleontology , fracture (geology)
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 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.