Twin prime editing (twinPE) uses two pegRNA–Cas9(H840A nickase)–reverse-transcriptase complexes that nick opposite strands of the target duplex, one upstream and one downstream. Each nicked 3' end primes reverse transcription off its own pegRNA template, writing a new single-stranded 3' flap that carries a shared homology sequence plus the new/edited bases:
Strand A (top): 5'---[nick A]-3' → RT → new flap A (homology + edit)
Strand B (bottom): 3'---[nick B]-5' → RT → new flap B (homology + edit, complementary)
Flaps anneal at the homology arm → heteroduplex → mismatch repair / replication → both strands carry the edit
Because the two flaps are complementary to each other (not to the original genomic strand they displace), resolution replaces the entire nick-to-nick interval — enabling precise insertions, deletions, and replacements far larger than single-flap prime editing (PE2/PE3) can reliably install, without a double-strand break.
This simulator scores each attempt with a simplified efficiency model combining three real determinants from the twinPE literature (Anzalone et al. 2022):
flapLen = insertSize + homology
homologyFactor = 1 - e^(-homology / 13) (saturates near the ~13-14 nt optimum)
rtFactor = e^(-flapLen / (40 · RT)) (longer flaps / weaker RT → more dropout)
spacingFactor = e^(-max(0, spacing-60)/80) (very wide nick spacing is harder to bridge)
efficiency = 55% · homologyFactor · rtFactor · spacingFactor
- Homology arm — the shared complementary stretch the two flaps use to anneal; too short and they never find each other, too long adds little.
- Insert / replacement size — how many bp of new sequence the flaps carry; larger edits mean a longer flap for the RT to finish.
- Nick-to-nick spacing — the genomic distance between the two nicks, i.e. the interval being replaced. This 2D view stretches the drawn duplex to always fully contain the current spacing, so both nick markers stay on-strand at every slider value.
- PE2 / PE2max / PEmax — editor variants with increasing RT processivity and evasion of mismatch-repair reversion, each attempt is drawn against the predicted efficiency.
Real-world relevance: twinPE (and its recombinase-extended cousin, PASTE) is used to install kilobase-scale insertions and gene-sized cassettes at a defined genomic locus without relying on double-strand breaks or donor-DNA HDR.