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Quadruple pegRNA enables programmable and efficient large genomic insertion

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Why This Matters

The development of quadruple pegRNA technology marks a significant advancement in genome editing, enabling precise, programmable, and efficient large DNA insertions without relying on double-strand breaks or donor DNA. This innovation has the potential to revolutionize gene therapy, functional genomics, and personalized medicine by making large-scale genetic modifications safer and more accessible for both research and clinical applications.

Key Takeaways

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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 in mice. Cell 188, 4275–4294.e23 (2025).

Gillmore, J. D. et al. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N. Engl. J. Med. 385, 493–502 (2021).

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