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. 2017 Nov 20;8:1610. doi: 10.1038/s41467-017-01732-9

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Conformational dynamics of target-bound dCas9:trRNA:crXNA complexes. a Single-molecule FRET experimental design. Surface-immobilised Cy3-labelled (green) DNA containing the AAVS1 target site (orange) and PAM site (white) binds Cy5-labelled (red) dCas9 complex (blue) to yield a FRET signal. b Representative single-molecule FRET trajectories show dCas9–crRNA complex binding in heteroduplex conformation as an increase to high FRET (top) through a transient intermediate (arrow). Irreversible conformational change into a dynamic intermediate FRET state (bottom). Corresponding time-binned FRET histograms (right), and Gaussian fits (black curves). Trajectories are 5 point averaged. c Time-binned FRET histograms for each guide: crRNA (red, n = 91); crHyb (grey, n = 57); crD.Hyb (magenta, n = 197), and crR.Hyb (green, n = 172)