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Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage.

Publication ,  Journal Article
Josephs, EA; Kocak, DD; Fitzgibbon, CJ; McMenemy, J; Gersbach, CA; Marszalek, PE
Published in: Nucleic acids research
October 2015

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.

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Published In

Nucleic acids research

DOI

EISSN

1362-4962

ISSN

0305-1048

Publication Date

October 2015

Volume

43

Issue

18

Start / End Page

8924 / 8941

Related Subject Headings

  • RNA
  • Protein Binding
  • Microscopy, Atomic Force
  • Humans
  • Endodeoxyribonucleases
  • Developmental Biology
  • DNA Cleavage
  • DNA
  • CRISPR-Associated Proteins
  • Binding Sites
 

Citation

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Josephs, E. A., Kocak, D. D., Fitzgibbon, C. J., McMenemy, J., Gersbach, C. A., & Marszalek, P. E. (2015). Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic Acids Research, 43(18), 8924–8941. https://doi.org/10.1093/nar/gkv892
Josephs, Eric A., D Dewran Kocak, Christopher J. Fitzgibbon, Joshua McMenemy, Charles A. Gersbach, and Piotr E. Marszalek. “Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage.Nucleic Acids Research 43, no. 18 (October 2015): 8924–41. https://doi.org/10.1093/nar/gkv892.
Josephs EA, Kocak DD, Fitzgibbon CJ, McMenemy J, Gersbach CA, Marszalek PE. Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic acids research. 2015 Oct;43(18):8924–41.
Josephs, Eric A., et al. “Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage.Nucleic Acids Research, vol. 43, no. 18, Oct. 2015, pp. 8924–41. Epmc, doi:10.1093/nar/gkv892.
Josephs EA, Kocak DD, Fitzgibbon CJ, McMenemy J, Gersbach CA, Marszalek PE. Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic acids research. 2015 Oct;43(18):8924–8941.
Journal cover image

Published In

Nucleic acids research

DOI

EISSN

1362-4962

ISSN

0305-1048

Publication Date

October 2015

Volume

43

Issue

18

Start / End Page

8924 / 8941

Related Subject Headings

  • RNA
  • Protein Binding
  • Microscopy, Atomic Force
  • Humans
  • Endodeoxyribonucleases
  • Developmental Biology
  • DNA Cleavage
  • DNA
  • CRISPR-Associated Proteins
  • Binding Sites