Supplementary Figure 1: FRET probe locations for DNA interrogation by Cas9–RNA and determination of Kd between Cas9–RNA and DNA. | Nature Structural & Molecular Biology

Supplementary Figure 1: FRET probe locations for DNA interrogation by Cas9–RNA and determination of Kd between Cas9–RNA and DNA.

From: Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis

Supplementary Figure 1

(a) Schematic of Cas9-RNA-DNA complex. The hybridized crRNA and tracrRNA are referred to as guide-RNA. Sequences in red denote guide sequence of the guide-RNA and the matching sequence of the DNA. The target strand is complementary to the guide-RNA. The non-target strand contains the PAM (5’-NGG-3’). A 22 nt biotin-labeled adaptor strand was used for surface immobilization of DNA and is highlighted in grey. (b) Cy3 and Cy5 labeling locations in Cas9-RNA-DNA complex (PDB ID: 4UN3)(Anders et al., Nature. 513, 569-573, 2014). (c) E histograms for cognate DNA and DNA with nPD=11 for dCas9, deCas9 and dCas9-HF1 vs. [Cas9-RNA] obtained in smFRET DNA interrogation experiments. (d-e) The apparent bound fraction vs. [Cas9-RNA] and fits for K d estimation. The number of PAM-distal mismatches (nPD) and PAM-proximal mismatches (nPP) are shown in cyan and orange respectively.

Back to article page