Prime Editing pegRNA Flap Equilibrium

pegRNA Design

Primer-binding site (PBS) 13 nt

RT template / edit length 10 nt

Run

Set pegRNA parameters, then run an editing cycle.

Live Statistics

Predicted efficiency

—

Trials run

Edited

Indel

How it works

Prime editing nicks only one DNA strand, then uses a prime-editing guide RNA (pegRNA) to template new genetic information directly onto the exposed 3′ end — no double-strand break, no donor template.

Nick + prime. A Cas9 nickase (H840A) nicks the PAM strand. The pegRNA's primer-binding site (PBS) hybridizes to the freed 3′ end, priming reverse transcriptase.
RT extension. Reverse transcriptase copies the pegRNA's RT template, extending a new 3′ flap that carries the edit — displacing the original, unedited 5′ flap.
Flap equilibrium. Cellular flap endonucleases (FEN1) and ligases resolve the branched intermediate. Preferential excision of the 5′ flap and ligation of the 3′ flap fixes the edit; the reverse outcome reverts to wild type.
PE3. Nicking the *non*-edited strand biases mismatch repair toward using the edited strand as template, roughly tripling efficiency — at the cost of occasional simultaneous double-strand nicks that repair as small indels.

This simulator scores each cycle with a simplified but literature-consistent model:

p_hybrid  = exp( -(PBS-13)^2 / 32 )        // Tm optimum near 13 nt
p_flap    = exp( -RT_len / 22 )            // longer edits excise less efficiently
p_edit    = min(0.95, 0.9 · p_hybrid · p_flap · (PE3 ? 1.55 : 1))
p_indel   = min(0.35, 0.015 + 0.0025·RT_len + (PE3 ? 0.045 : 0))
p_revert  = 1 − p_edit − p_indel

Real prime editors (PE2/PE3, Anzalone et al. 2019) show the same qualitative trends: an optimal PBS length around 12–14 nt, falling efficiency for longer insertions/edits, and a PE3 efficiency boost paired with a modest indel cost — the exact percentages here are illustrative, not measured values.

Genomic DNA pegRNA 3′ edited flap 5′ original flap

⚠ Couldn't load the 3D engineThree.js failed to load from the CDN. Check your connection and reload.