Fig. 5: Metastable states control the targeting dynamics.
a) A coarse-grained version of the reaction scheme shown in Fig. 1a. Apart from the unbound and post-cleavage state, the targeting-reaction pathway is reduced to just three states: PAM bound and R-loop closed (0 bp hybrid), intermediate R-loop (7–13 np hybrid), and open R-loop (20 bp hybrid). b Microscopic free-energy landscape for the on-target exposed to 1 nM (d)Cas9-sgRNA (Fig. 4a) with coarse-grained states and rates indicated in black. c The calculated (see Methods) coarse-grained forward and backward rates on the on-target. Purple triangles are rates from Ivanov et al.42, when available at zero torque. d Microscopic free-energy landscape for an off-target with a mismatch at position 3 (blue), together with the on-target free-energy landscape (pink). Red arrow indicates the free-energy penalty \(\delta {{\epsilon }}_{3}\) at the mismatch, and black arrow indicates the resulting shift in barrier height. e The calculated coarse-grained forward and backward rates on an off-target with a mismatch at position 3. Orange arrow highlights the rate that changed considerably compared to on-target. Purple triangles are rates from Ivanov et al.42, when available at zero torque. f Microscopic free-energy landscape for an off-target with a mismatch at position 15 (blue), together with the on-target free-energy landscape (pink). Red arrow indicates free-energy penalty \(\delta {{\epsilon }}_{15}\) at the mismatch, and black arrow indicates the resulting shift in barrier height. g The calculated coarse-grained forward and backward rates on an off-target with a mismatch at position 15. Orange arrow highlights the rate that changed considerably compared to on-target. In Fig. 5c, e, and g, central line represents the median, the box plots represent the interquartile range, and whiskers represent the full range among our 33 near equivalent solutions.
