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.
Highlights
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas[9] from Streptococcus pyogenes along with a single guide RNA has emerged as a versatile toolbox for genome editing
To investigate the mechanisms underlying Cas9-catalyzed cleavage process, here, we present the first computational simulation study on the Cas9-single guide RNA (sgRNA)-double-stranded DNA (dsDNA) system at the atomic level to address the following two questions: 1) Could the Mg2+ ions bridge the distance gap between the active sites of Cas[9] and ntDNA? 2) Where is the cleavage site on ntDNA? Our simulation results demonstrate that introduction of two Mg2+ ions at −4P remarkably induces conformation changes to bring the active site of the RuvC domain and ntDNA into proximity, forming structurally and energetically favorable coordination in between the catalytic residues and the scissile phosphate for cleavage reaction to occur
Ion was placed in the HNH domain on the basis of the one-metal ion catalysis mechanism (Fig. 1C), whereas two Mg2+ ions were positioned at the interface of the RuvC domain and the ntDNA at various positions according to the two-metal-ion mechanism (Fig. 1D)[17,18,19]
Summary
The CRISPR-associated endonuclease Cas[9] 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 remains controversial whether Cas[9] generates blunt-ended or staggeredended 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. We used molecular dynamics simulations to search for the active state and to test our hypothesis
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