Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9

Digvijay Singh, Samuel H. Sternberg, Jingyi Fei, Jennifer A. Doudna () and Taekjip Ha ()
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Digvijay Singh: Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign
Samuel H. Sternberg: University of California
Jingyi Fei: University of Illinois at Urbana-Champaign
Jennifer A. Doudna: University of California
Taekjip Ha: Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign

Nature Communications, 2016, vol. 7, issue 1, 1-8

Abstract: Abstract Binding specificity of Cas9–guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9–RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from 2 s−1 upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate

Date: 2016
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DOI: 10.1038/ncomms12778

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