CRISPR-Cas9 Target Search (2D)

Guide RNA Design

Mismatches vs. target 0 / 20

Place mismatches in PAM-proximal seed region

Scan speed 55 nt/s

Repair Pathway (on cut)

Live Readout

Stage

Scanning

Cleavage efficiency

100%

Search cycles

Successful edits

Success rate

0.0%

Spacer match

20/20

Last outcome

—

How it works

Cas9 slides continuously along a 100‑nt DNA window, pausing at every candidate PAM (5′-NGG-3′). Most are decoys and get rejected immediately; only the one preceded by a spacer that can base-pair with the loaded guide RNA opens an R-loop and gets fully interrogated, 20 nucleotides at a time.

Each nucleotide position along the spacer carries a different weight — positions closest to the PAM (the "seed") are far more sensitive to a mismatch than the distal end:

efficiency = exp( -Σ w(i) )   for every mismatched position i
w(i) ≈ 0.62 near PAM (seed)  …  0.05 far from PAM

The "Place mismatches in seed region" toggle moves your chosen mismatches between the two ends of the spacer without changing their count, so you can see the same number of errors produce a very different cleavage probability depending only on where they land.

Scanning / PAM check — Cas9's continuous 1D search along the DNA, pausing at every NGG.
R-loop / interrogation — the helix opens at the real target and each spacer base is checked against the guide RNA in turn (green = match, red = mismatch).
Cleavage roll — on reaching the end of the spacer, a random draw against the current efficiency decides whether the double-strand break actually happens.
Repair — a successful cut is closed by whichever pathway is selected: NHEJ (fast, produces a random indel) or HDR (slow, produces an exact correction).

A T G C Cas9 complex