Supplementary Figure 9: smFRET unwinding experiments using pre-unwound DNA and probe locations for smFRET assay to investigate Cas9–RNA-induced DNA unwinding in PAM-proximal site.
From: Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis
(a) Schematics of smFRET DNA unwinding experiments utilizing pre-unwound DNA targets as described in Fig. 5c. (b-d) E histograms vs nPD (left) and representative single molecule time traces of donor and acceptor intensities (middle) and idealized E values (right). (e) Locations of EngCas9 mutations in dCas9-RNA-DNA complex (PDB ID: 4UN3)(Anders et al., Nature. 513, 569-573, 2014) (f) Schematics of smFRET DNA unwinding experiments to investigate Cas9-RNA induced DNA unwinding in PAM-proximal site (g) Probe locations for smFRET experiments to investigate Cas9-RNA induced DNA unwinding in PAM-proximal site, mapped to the structure of Cas9-RNA-DNA complex (PDB ID: 5F9R(Jiang et al., Science. 351, 867-871, 2016)).
