CRISPR-Cas9 R-Loop Zipper Kinetics

Cas9 does not check its 20-nucleotide guide RNA against target DNA all at once — it zips the R-loop open base pair by base pair, starting at the PAM and working outward, and the reaction can reverse at any step. This simulator renders that zippering process in 3D as a biased random walk with a position-dependent mismatch penalty: the walk stalls hard when a mismatch sits in the PAM-proximal seed region but barely notices one near the far end of the protospacer, which is exactly why some off-target sites with distal mismatches get cut almost as efficiently as the intended target while single seed mismatches can abolish cleavage entirely. Place a mismatch cluster anywhere along the guide, choose a canonical or suboptimal PAM, run single animated binding trials or a fast batch of 100, and compare the exact theoretical cleavage probability against the empirical success rate.