Although therapeutic genome editing holds great potential to remedy diverse inherited and acquired disorders, targeted installation of medium-to-large genomic modifications in therapeutically relevant cells remains challenging.
Here we develop prime assembly, an approach that permits DNA sequence assembly and integration in human cells leveraging CRISPR-targeted dual flap synthesis.
This method enables RNA-programmable site-specific integration of single or double-stranded DNA fragments.
Unlike homology-directed repair, prime assembly is similarly active in dividing and non-dividing cells.
We applied prime assembly to perform targeted exon recoding, transgene integration and megabase-scale rearrangements, including at therapeutically relevant loci in primary human cells.
Prime assembly expands the capabilities of genome engineering by enabling the targeted integration of medium to large-sized DNA sequences without relying on double-stranded DNA donors, nuclease-driven double-strand breaks or cell cycle progression.
Nature 2026 Sep 16.
