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Nat Commun. 2016 Sep 14;7:12778. doi: 10.1038/ncomms12778.

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

Singh D1, Sternberg SH2, Fei J3,4, Doudna JA2,5,6,7,8, Ha T1,3,4.

Author information

1
Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
2
Department of Chemistry, University of California, Berkeley, California 94720, USA.
3
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
4
Howard Hughes Medical Institute, Baltimore, Maryland 21205, USA.
5
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
6
Howard Hughes Medical Institute, Berkeley, California 94720, USA.
7
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
8
Innovative Genomics Initiative, University of California, Berkeley, California 94720, 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 <0.006 s(-1) to >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 <0.006 s(-1)), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

Conflict of interest statement

S.H.S and J.A.D. are inventors on a related patent application. The other authors declare no competing financial interests.

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