title: Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage.
abstract: CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single 'guide RNA' molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a 'conformational gating' mechanism driven by the interactions between the guide RNA and the 14th-17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex.
authors.affiliation: Department of Mechanical Engineering and Materials Science, Edmund T. Pratt, Jr. School of Engineering, Duke University, Durham, NC 27708, USA eaj20@duke.edu.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Josephs
authors.affiliation: Department of Biomedical Engineering, Edmund T. Pratt, Jr. School of Engineering, Duke University, Durham, NC 27708, USA.
authors.forename: D Dewran
authors.initials: DD
authors.lastname: Kocak
authors.affiliation: Department of Mechanical Engineering and Materials Science, Edmund T. Pratt, Jr. School of Engineering, Duke University, Durham, NC 27708, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fitzgibbon
authors.affiliation: Department of Biomedical Engineering, Edmund T. Pratt, Jr. School of Engineering, Duke University, Durham, NC 27708, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: McMenemy
authors.affiliation: Department of Biomedical Engineering, Edmund T. Pratt, Jr. School of Engineering, Duke University, Durham, NC 27708, USA Center for Genomic and Computational Biology, Duke University, Durham, NC 27708, USA Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Gersbach
authors.affiliation: Department of Mechanical Engineering and Materials Science, Edmund T. Pratt, Jr. School of Engineering, Duke University, Durham, NC 27708, USA pemar@duke.edu.
authors.forename: Piotr E
authors.initials: PE
authors.lastname: Marszalek
mesh_terms: D001665:Binding Sites; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D053837:DNA Cleavage; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D012313:RNA; D004247:DNA; D004706:Endodeoxyribonucleases