Prime assembly enables targeted integration of large DNA sequences in human cells

Researchers have developed "prime assembly," a new genome editing technique that enables targeted integration of medium-to-large DNA sequences in human cells without requiring double-strand breaks or cell cycle progression. The method supports exon recoding, transgene insertion, and megabase-scale rearrangements in therapeutically relevant cells. Prime assembly leverages CRISPR-targeted dual flap synthesis to integrate single or double-stranded DNA fragments, functioning effectively in both dividing and non-dividing cells unlike homology-directed repair. The approach was validated in primary human cells, including CD34+ hematopoietic stem and progenitor cells and CD3+ T cells, demonstrating potential for therapeutic applications. The technique was tested across multiple cell lines including K562, Jurkat, and HEK293T cells, with various donor formats and concentrations optimized for different fragment sizes. The researchers also developed analytical methods including amplicon sequencing, nanopore sequencing, and Donor-seq to characterize integration outcomes and off-target effects.