Same birth-death zipper model as the 3D version, drawn two different ways instead of as a rotating helix. R-loop extension x runs 0…20 as a biased random walk: forward (pairing) rate kf, backward (unpairing) rate kb per base pair.
matched bp: k_f = k0, k_b = kb0
mismatched bp: k_f = k0·e^(−B(i)), k_b = kb0·e^(B(i))
B(i) = Bmax·[0.15 + 0.85·e^(−(i−1)/6)] (i = distance from PAM)
Top panel — a free-energy landscape built directly from the same rate constants: each step's height change is ΔG(i) = ln(kb(i)/kf(i)), so a mismatch near the PAM (small i) draws a steep uphill wall right at the start of the walk, while the identical mismatch near the distal end barely dents the landscape — the landscape shape is the mechanistic reason for the seed-vs-distal asymmetry.
Bottom panel — a kinetic race: many independent stochastic realizations of the exact same walk plotted as extension-vs-step traces (a "spaghetti plot"), green if they reach bp 20 before bp 0, red otherwise. The fraction of green traces converges to the exact absorption probability computed analytically from the birth-death branching ratios, shown as "Model P(cleave)".
- Mismatch position — where along the 20-nt protospacer (counted from the PAM) the mismatch cluster starts.
- Mismatch cluster length — how many consecutive mismatched bases sit there (0 = perfect match, positive control).
- PAM quality — a suboptimal NAG PAM lowers the initial engagement probability, scaling predicted off-target frequency down independently of the R-loop kinetics.