Collectively, prime assembly enables the targeted integration of medium to large DNA sequences in non-dividing cells without relying on nuclease-driven double strand breaks, or double-stranded DNA donors. Our study establishes in cellulo DNA assembly as a new modality for genome editing.

Finally, using a CDK4/6 inhibitor and cell cycle analysis, we observed efficient PA in cells arrested in G1.

Under selective pressure, prime assembly enables the assembly of up to four ssDNA fragments, and the installation of megabase-scale rearrangements.

Using ssDNA donors, we achieved targeted transgene integration at two loci in CD34+ hematopoietic stem and progenitor cells from three different healthy donors.

Using this approach, we achieved targeted exon recoding and transgene integration with efficiencies of up to 50% and 25%, respectively.

Taking advantage of CRISPR-targeted flap synthesis, we developed an approach to assemble and integrate double-stranded or single-stranded DNA donors at specific genomic loci. We named this method prime assembly.