QuadPE—a quadruple paired prime editing guide RNA strategy—enables efficient and programmable insertion of large DNA fragments.

Precise, site-specific insertion of large gene sequences holds great promise for the treatment of diverse genetic disorders. Although prime editing using paired guide RNAs (pegRNAs) can mediate targeted integration, insertion efficiency drops sharply for payloads exceeding 300 base pairs1–3. Here we present a rationally designed quadruple pegRNA strategy (QuadPE) for efficient and programmable insertion of large DNA fragments. Through screening different designs, we identified that combinations of two genome-targeting pegRNAs in a PAM-out or PAM-in orientation, when paired with two donor-targeting pegRNAs in linear or circular form, yield optimal efficiency. Using QuadPE, we achieved stable integration efficiency of DNA fragments ranging from 1.6 to 26 kb, with efficiencies of around 40% at multiple loci with minimal off-target insertion activity. QuadPE substantially outperformed recombinase-mediated (PASSIGE and PASTE)4,5 and transposase-mediated (CAST)6 insertion systems,

Join Xtalks for an insightful webinar focusing on enhanced pegRNA strategies that improve editing efficiency in genome engineering, without increasing complexity.

This post provides tips for designing a prime editing experiment, from pegRNA design, PE tool selection, and choice of DNA sequence to edit.

The advances in editing efficiency and delivery are important steps toward in vivo therapeutic prime editing.

Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2022). Wang, J. et al. Efficient targeted insertion of large DNA fragments without DNA donors. Nat. Methods 19, 331–340 (2022). Yarnall, M. T. N. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat. Biotechnol. 41, 500–512 (2023). Pandey, S. et al. Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing. Nat. Biomed. Eng. 9, 22–39 (2025). Witte, I. P. et al. Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase. Science 388, eadt5199 (2025). Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016). Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017). Sousa, A. A. et al. In vivo prime editing rescues alternating hemiplegia of childhood...

The prime editors (PEs) have shown great promise for precise genome modification. Here the authors place a stabilizing viral xrRNA motif to the 3′ of pegRNAs to enhance editing efficiencies.

Read the full paper: Disrupting pegRNA intramolecular complementarity via PBS and spacer sequence alterations can enhance prime editing efficiency

Because a low intracellular concentration of synthetic pegRNA after PE translation may limit prime editing following LNP delivery, we initially sought to compare LNP-mediated prime editing efficiencies using either a synthetic HM-pegRNA, as previously described22, or a synthetic epegRNA containing a 3′ pseudoknot motif that protects the pegRNA from degradation24. To deliver prime editing components via LNPs, we used microfluidic mixing to separately formulate nucleoside-modified mRNA encoding PEmax, (e)pegRNA and nicking gRNA (ngRNA) into OF-02 LNPs (Fig. 1a). The OF-02 LNP formulation was chosen for its ability to efficiently deliver mRNA to the liver28. Although we considered coformulating the (e)pegRNA and ngRNA owing to their similar size and structure, we chose to formulate them separately to provide better control of individual doses and streamline the formulation process when testing different combinations of mRNA, (e)pegRNA and ngRNA. We admixed LNPs individually containing the editor mRNA, (e)pegRNA or ngRNA immediately before either treating Hepa1-6 cells with increasing doses of PE-LNPs or retro-orbitally (RO) administering 2 mg kg−1 PE-LNPs to adult C57BL/6 mice. The LNPs were mixed at an initial ratio of 1:0.9:0.1 mRNA:(e)pegRNA:ngRNA by total RNA mass based on previous work demonstrating efficient editing in vitro with this ratio29. Using a previously...

Lushington and colleagues synthesize the rapidly evolving prime editing landscape, from pegRNA and editor engineering to dual-pegRNA and integrase/recombinase platforms, and connect these advances to safety frameworks and the first in-human study. The review provides a clinically oriented roadmap for translating precision genome rewriting into therapies.

Prime editing is a genome engineering tool that allows installation of small edits with high precisi

This post provides tips for designing a prime editing experiment, from pegRNA design, PE tool selection, and choice of DNA sequence to edit.

Engineered deletions are a powerful probe for studying genome architecture, function, and regulation. Yet, the lack of effective methods to create them in large numbers and at multi-kilobase scale has...

a, Schematic of the PE protein (ePPEplus in this study) and pegRNA expression vector used for prime editing. nSpCas9, SpCas9 nickase. M-MLV RT, Moloney murine leukemia virus reverse transcriptase (RNase H domain deleted). NC, viral nucleocapsid protein. CaMVt, cauliflower mosaic virus polyadenylation signal terminator. NLS, nuclear localization signal. NLSc−Myc, the c-Myc NLS. NLSSV40, the SV40 NLS. bpNLSSV40, the bipartite SV40 NLS. vbpNLSSV40, a variant of the bipartite SV40 NLS. p35C, en35S (35S enhancer)-CmYLCV-AtU6 composite promoter. tOsGly, tRNAOsGly. HDV, hepatitis delta virus ribozyme. HSP18.2t, Arabidopsis heat shock protein 18.2 terminator. b, Schematic for gene knockout using single-pegRNA prime editing strategies, including single-base insertion, base substitutions to create a TAA stop codon, and insertion of a TAA stop codon adjacent to an intact codon. The schematic was created using items (Created in BioRender. Gao, C. (2026). https://BioRender.com/mq5dexa) from BioRender and subsequently refined with Adobe Illustrator. c, Frequencies of desired substitutions (sub) and insertions (ins) for gene knockout at six rice target sites (three biological replicates, n = 3) using the ePPEplus and single-pegRNA systems. d, Average frequencies of desired edits across all six rice targets (three biological replicates, n = 18) in c. e, RNA secondary structures and folding stabilities (ΔG) of...