Publications [#322156] of Piotr E. Marszalek

Papers Published

1. 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, vol. 43 no. 18 (October, 2015), pp. 8924-8941 [doi]
   (last updated on 2023/06/01)

   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.