Figure 1: Cas9–RNA binding to a cognate sequence.
From: Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9
(a) Schematic of single-molecule FRET assay. High-FRET signal resulted when Cas9 in complex with an acceptor (Cy5)-labelled guide-RNA (Cas9–RNA) bound a surface-immobilized, donor (Cy3)-labelled target DNA that contains the cognate sequence (red DNA segment) and PAM (yellow segment). (b) A representative smFRET time trajectory of a stably bound Cas9–RNA in the presence of 20 nM Cas9–RNA in solution. (c) FRET histograms obtained with cognate DNA (top) and negative controls with a non-cognate DNA (middle) and with RNA only (without Cas9; bottom). The number of molecules included ranged from 568 to 1,314. Corresponding images of donor and acceptor channels are shown. (d) A representative smFRET time trajectory of real-time binding of Cas9–RNA in a single step after 20 nM Cas9–RNA is added at the time point indicated.
