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

Zhicheng Zuo, Jin Liu

Research output: Contribution to journalArticle

24 Citations (Scopus)

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.

Original languageEnglish
Article number37584
JournalScientific Reports
Volume5
DOIs
StatePublished - 22 Nov 2016

Fingerprint

Guide RNA
Molecular Dynamics Simulation
Clustered Regularly Interspaced Short Palindromic Repeats
Ions
Catalytic Domain
DNA

Cite this

@article{33a49e1b63974a1481974d6cf6f1228c,
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.",
author = "Zhicheng Zuo and Jin Liu",
year = "2016",
month = "11",
day = "22",
doi = "10.1038/srep37584",
language = "English",
volume = "5",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",

}

Cas9-catalyzed DNA Cleavage Generates Staggered Ends : Evidence from Molecular Dynamics Simulations. / Zuo, Zhicheng; Liu, Jin.

In: Scientific Reports, Vol. 5, 37584, 22.11.2016.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Cas9-catalyzed DNA Cleavage Generates Staggered Ends

T2 - Evidence from Molecular Dynamics Simulations

AU - Zuo, Zhicheng

AU - Liu, Jin

PY - 2016/11/22

Y1 - 2016/11/22

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84996524306&partnerID=8YFLogxK

U2 - 10.1038/srep37584

DO - 10.1038/srep37584

M3 - Article

C2 - 27874072

AN - SCOPUS:84996524306

VL - 5

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 37584

ER -