Figure 3: Cas9–RNA-bound state lifetimes for different DNA targets.
From: Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9
(a) smFRET time trajectory (donor and acceptor intensities, top, and idealized FRET via hidden Markov modelling (HMM) analysis, bottom) for 9–20mm DNA target in the presence of 20 nM Cas9–RNA. Reversible Cas9–RNA association to high- and mid-FRET states and disassociation to zero-FRET state are shown. (b) Transition density plots show relative transition frequencies between different FRET states for 9–20mm and 5–20mm DNA targets. [Cas9–RNA]=20 nM. (c) The amplitude-weighted lifetime, τavg, of the putative bound state, lifetime of high to zero and mid to zero FRET state transitions and biomolecular rate association constants for different DNA targets. On the basis of our model, the mid- and high-FRET states correspond to sampling and RNA–DNA heteroduplex modes, respectively. (d) Lifetime comparison of DNA targets with the respective DNA targets containing mismatches after the roadblock. All the data shown in the figure are from independent experiments and error bars represent s.d. for n=3 (n=2 for few sets).
