Show simple item record

dc.contributor.authorRicci, Clarisse G.
dc.contributor.authorChen, Janice S.
dc.contributor.authorMiao, Yinglong
dc.contributor.authorJinek, Martin
dc.contributor.authorDoudna, Jennifer A.
dc.contributor.authorMcCammon, J. Andrew
dc.contributor.authorPalermo, Giulia
dc.date.accessioned2020-12-21T19:53:53Z
dc.date.available2020-12-21T19:53:53Z
dc.date.issued2019-03-07
dc.identifier.citationClarisse G. Ricci, Janice S. Chen, Yinglong Miao, Martin Jinek, Jennifer A. Doudna, J. Andrew McCammon, and Giulia Palermo ACS Central Science 2019 5 (4), 651-662 DOI: 10.1021/acscentsci.9b00020en_US
dc.identifier.urihttp://hdl.handle.net/1808/30983
dc.description.abstractCRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations—using Gaussian accelerated MD (GaMD)—are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at distal sites with respect to the protospacer adjacent motif (PAM) can induce an extended opening of the RNA:DNA heteroduplex, which leads to newly formed interactions between the unwound DNA and the L2 loop of the catalytic HNH domain. These conserved interactions constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and hampering its activation for cleavage. Remarkably, depending on their positions at PAM distal sites, DNA mismatches responsible for off-target cleavages are unable to “lock” the HNH domain, thereby leading to the unselective cleavage of DNA sequences. In consistency with the available experimental data, the ability to “lock” the catalytic HNH domain in an inactive “conformational checkpoint” is shown to be a key determinant in the onset of off-target effects. This mechanistic rationale contributes in clarifying a long lasting open issue in the CRISPR-Cas9 function and poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.en_US
dc.publisherAmerican Chemical Societyen_US
dc.rightsCopyright © 2019 American Chemical Societyen_US
dc.subjectPolyacrylamideen_US
dc.subjectGeneticsen_US
dc.subjectGenomicsen_US
dc.subjectCrystal cleavageen_US
dc.subjectNucleic acid structureen_US
dc.titleDeciphering Off-Target Effects in CRISPR-Cas9 through Accelerated Molecular Dynamicsen_US
dc.typeArticleen_US
kusw.kuauthorMiao, Yinglong
kusw.kudepartmentCenter for Computational Biologyen_US
kusw.kudepartmentMolecular Biosciencesen_US
dc.identifier.doi10.1021/acscentsci.9b00020en_US
dc.identifier.orcidhttps://orcid.org/0000-0002-3289-2248en_US
dc.identifier.orcidhttps://orcid.org/0000-0003-3714-1395en_US
dc.identifier.orcidhttps://orcid.org/0000-0001-9161-999Xen_US
dc.identifier.orcidhttps://orcid.org/0000-0003-3065-1456en_US
dc.identifier.orcidhttps://orcid.org/0000-0003-1404-8737en_US
kusw.oaversionScholarly/refereed, publisher versionen_US
kusw.oapolicyThis item meets KU Open Access policy criteria.en_US
dc.rights.accessrightsopenAccessen_US


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record