Figure EV2. Stoichiometry of sgRNA binding to Cas9.

1. Fluorescence emission spectra of 20 nM fluorescent Cas9 (D435C–E945C) excited at 532 nm. The Cy3 and Cy5 fluorescence intensity changes were coupled with the FRET efficiency change, according to the sgRNA concentration.
2. Quantification of the ratio between Cy3 and Cy5 fluorescence intensities. The ratios of Cy5 fluorescence peak intensity over Cy3 fluorescence peak intensity were plotted against the sgRNA concentration (n = 3 for each sgRNA concentration). The FRET efficiency change coupled with the sgRNA binding was almost saturated at the Cas9 to sgRNA ratio of 1:1.