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

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  • Digvijay Singh

    (Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign
    Present address: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
    Present address: Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21205, USA
    Present address: Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21205, USA)

  • Samuel H. Sternberg

    (University of California)

  • Jingyi Fei

    (University of Illinois at Urbana-Champaign
    Howard Hughes Medical Institute
    Present address: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA)

  • Jennifer A. Doudna

    (University of California
    University of California
    Howard Hughes Medical Institute
    Lawrence Berkeley National Laboratory)

  • Taekjip Ha

    (Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign
    University of Illinois at Urbana-Champaign
    Howard Hughes Medical Institute
    Present address: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA)

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

Suggested Citation

  • Digvijay Singh & Samuel H. Sternberg & Jingyi Fei & Jennifer A. Doudna & Taekjip Ha, 2016. "Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9," Nature Communications, Nature, vol. 7(1), pages 1-8, November.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms12778
    DOI: 10.1038/ncomms12778
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    Cited by:

    1. Bin Yang & Haonan Wang & Jilie Kong & Xueen Fang, 2024. "Long-term monitoring of ultratrace nucleic acids using tetrahedral nanostructure-based NgAgo on wearable microneedles," Nature Communications, Nature, vol. 15(1), pages 1-15, December.

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