Prime assembly integrates DNA fragments up to 6.5 kb without viral vectors
Researchers at Seoul National University developed prime assembly, a method that uses prime editors to create complementary 3'-flaps on genomic and donor DNA, allowing megabase-scale genomic excision and kilobase-scale donor insertion. The technique, published in Nature Biotechnology on 26 January 2025, accepts DNA plasmids and linear double-stranded DNA as donors, ranging from 1.0 to 6.5 kb in size. It replaced endogenous sequences with a 2.9-kb donor DNA fragment at up to 57.8% efficiency in HEK293T cells, with over 90% accuracy for integrated PA fragments. In primary human T cells, it enabled site-specific chimeric antigen receptor integration with up to 28.1% efficiency. In mice, hydrodynamic injection achieved 4.3% average integration efficiency in GFP-positive hepatocytes.
The method may enable therapeutic gene replacement in primary cells without relying on viral vectors.
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