Standard prime editing is wonderful for swapping a few nucleotides. It is poor at inserting whole genes. The 3′ extension of a pegRNA can hold maybe 50 base pairs of new sequence — enough to fix a point mutation or add a short tag, nowhere near enough to insert a full coding sequence for cystic fibrosis transmembrane conductance regulator (CFTR), dystrophin, or factor VIII. Twin prime editing and PASTE are the two-stage solutions to that problem. They combine prime editing with a second mechanism — paired editing in one case, serine integrases in the other — to deliver kilobase-scale payloads to defined genomic sites without ever creating a free double-strand break.
The primary keyword here is twin prime editing PASTE, and the broader story is the field's pivot toward "templated insertion" — depositing whole genes at the right address with the precision of CRISPR and the cargo capacity of viral vectors.
What Are Twin Prime Editing and PASTE?
Twin prime editing (twinPE) was introduced by Andrew Anzalone, David Liu, and colleagues in Nature Biotechnology in 2022 ("Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing"). The trick is to use two pegRNAs that target opposite strands of the same locus, each writing a small piece of new sequence. The resulting nicks and edits cooperate to install matched recombination sites — typically attB or attP — flanking a region of interest. After twin prime editing has installed these "landing pads," the genomic locus is now a substrate for follow-on operations.
PASTE — Programmable Addition via Site-specific Targeting Elements — was introduced by Anzalone, Feng Zhang, and colleagues in Nature Biotechnology in 2023. PASTE goes further: it fuses a prime editor directly to a serine integrase (Bxb1 or related). The prime editor first installs an attB landing pad at the target site; then the integrase recombines a donor plasmid carrying attP and a kilobase-scale payload into the landing site in a single workflow. The result is precise, site-specific insertion of cargoes up to roughly 36 kilobases — large enough for most human coding sequences.
PASTE is the technology behind Prime Medicine's PASSAGE platform, which the company has positioned as its solution for "one-and-done" replacement of disease genes regardless of which mutation a patient carries.
How They Work at the Molecular Level
Twin prime editing
1. Two pegRNAs target opposite strands in proximity at a single locus. Each pegRNA contains a spacer (for Cas9 targeting), a primer-binding site, and a reverse-transcriptase template encoding the new sequence to write.
2. The prime editor creates a nick on one strand, the reverse transcriptase extends from the primer-binding site, and a flap of new sequence is generated. This happens twice — once on each strand.
3. The two flaps are resolved by DNA repair, replacing the intervening sequence with the user-designed insert. The deleted region is cleanly removed and the new sequence — typically containing an attB site — is in place.
4. Optional integrase addition. Once the landing pad is installed, a serine integrase such as Bxb1 can deliver a kilobase-scale donor.
PASTE
PASTE collapses these steps into a single editing complex:
1. The fused prime editor + Bxb1 integrase complex visits the target site.
2. The prime editor writes an attB sequence into the genome via the same mechanism as standard prime editing.
3. The Bxb1 integrase, now positioned at the landing site, recombines with an attP-containing donor plasmid carrying the gene to be inserted.
4. The donor is integrated at the landing site as a precise, large insertion — up to ~36 kb in the original 2023 paper.
The whole reaction is enzymatic and avoids both double-strand breaks and reliance on host HDR machinery — a major safety and efficiency advantage in cells that do not divide (neurons, muscle, hepatocytes).
Key Papers and Milestones
- Anzalone et al., 2019 (Nature). Original prime editing paper.
- Anzalone et al., 2022 (Nature Biotechnology). Twin prime editing for programmable deletion, replacement, integration, and inversion of large sequences.
- Yarnall, Anzalone, Zhang et al., 2023 (Nature Biotechnology). PASTE — prime editor fused to Bxb1 integrase for kilobase-scale insertions.
- Pandey et al., 2024. Improved PASTE efficiency in primary human hepatocytes.
- Prime Medicine's PASSAGE program disclosures, 2023–2026. Clinical preparations for liver-directed gene replacement using PASTE-based approaches.
- Tessera Therapeutics MPS (Mobile Programmable Sequences) work. A parallel platform leveraging mobile genetic elements and integrases for large-cargo delivery.
Applications and Use Cases
Cystic fibrosis (CFTR replacement). CFTR has more than 2,000 disease-causing mutations. Replacing the entire coding sequence in a single, mutation-agnostic edit is the holy grail. Prime Medicine's PASSAGE program is targeting CFTR with a PASTE-based approach, with a focus on lung-directed delivery.
Hemophilia. Inserting a functional factor VIII or factor IX gene into a safe-harbor or native locus. The advantage over AAV gene therapy is durability — integrated genes don't dilute as cells divide.
Lysosomal storage disorders. Many such diseases (MPS, Pompe, Fabry) are caused by single-gene loss of function and would benefit from precise replacement of a full coding sequence.
Friedreich's ataxia and muscular dystrophy. Both involve genes too large to be delivered easily by AAV. PASTE-style integration may eventually deliver dystrophin or frataxin in their full forms.
T-cell engineering. PASTE has been used in primary T cells to install large cargos at the TRAC locus — a route to safer next-generation CAR-T cell therapies.
Twin Prime Editing / PASTE vs Standard Prime Editing vs AAV Gene Addition
| Feature | Standard prime editing | Twin prime editing | PASTE | AAV gene addition |
|---|---|---|---|---|
| Cuts DNA? | Single nick | Two nicks | Two nicks + integrase | No |
| Max insert size | ~50 bp | ~few hundred bp practical | ~36 kb | ~4.7 kb (single AAV) |
| Site-specific? | Yes | Yes | Yes | Random or AAV ITR |
| Integration durability | Full | Full | Full | Episomal (fades) |
| Mutation-agnostic? | No (one edit at a time) | Possible | Yes | Yes |
| Stage | Approved program prep | Preclinical | Preclinical / IND-enabling | Multiple approved |
Connection to the Broader Gene Editing Ecosystem
Twin prime editing and PASTE extend the prime editing chassis built by Andrew Anzalone in David Liu's lab. They sit alongside bridge RNAs as the field's main strategies for site-specific large-cargo insertion — both leverage serine recombinases / integrases, though PASTE pairs them with a prime editor while bridge RNAs use bare RNA-guided recombinases. They share the delivery system constraints of all CRISPR tools, with the additional challenge that the donor DNA is itself large. PASTE is being commercialized by Prime Medicine, whose first clinical asset (PM359 for chronic granulomatous disease) is a standard prime editor — see our article on Casgevy for the broader picture of how the first generation of CRISPR therapies is reaching patients while these next-generation tools mature.
Current Limitations and Challenges
- Efficiency. PASTE integration in human cells remains lower than ideal — typically 5–50% in cell lines and lower in primary cells without optimization.
- Donor delivery. Large donors need a delivery vehicle. AAV maxes out around 4.7 kb; LNP-mRNA cannot easily carry plasmid donors. Dual-AAV and integrated donor cassettes are workarounds.
- Integrase off-target sites. Bxb1 attP sites are rare in the human genome but not absent — pseudo-attB sites can produce unintended integrations.
- Cargo capacity vs efficiency tradeoff. Larger inserts integrate less efficiently.
- Manufacturing complexity. A multi-component editor (prime editor + integrase + donor) is harder to produce and characterize than a single Cas9 RNP.
FAQ
What is the difference between twin prime editing and PASTE?
Twin prime editing uses two pegRNAs to install or delete sequences at a single locus, including landing pads for downstream integrase reactions. PASTE fuses a prime editor directly to a serine integrase, doing the landing-pad install and the integrase reaction in one architecture.
Who invented PASTE?
Andrew Anzalone (then at Prime Medicine), Feng Zhang (Broad Institute), and colleagues. The platform was published in Nature Biotechnology in 2023.
How large an insert can PASTE deliver?
Up to roughly 36 kilobases in the original 2023 publication, though real-world efficiency drops at the upper end of the range.
Is PASTE in clinical trials?
Not yet. Prime Medicine's first clinical assets use standard prime editing. PASTE-based programs (PASSAGE) are in IND-enabling work as of 2026.
Why is this better than AAV gene therapy?
Site-specific integration produces durable expression — the gene sits in chromosomal DNA, not as an episomal AAV that dilutes with cell division. It also allows insertion at the native locus, preserving regulatory context.
Can PASTE replace a whole disease gene regardless of which mutation a patient carries?
Yes — that is the central appeal. By installing a functional copy of the entire coding sequence, PASTE provides a "mutation-agnostic" approach that does not require a separate edit per allele.
