NatureApr 29, 2026

Using prime assembly with linear DNA to insert large genetic sequences

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

Prime assembly enabled targeted insertion of large DNA fragments without double-stranded DNA breaks, recombinases, or canonical homology-directed repair.

Evidence

This genome-engineering platform experiment used linear DNA donors overlapping twin prime-editing flaps to insert one or multiple fragments totaling 0.1 kb to 11 kb, including in non-cycling cells.

Caveat

The abstract reports platform performance and mechanistic features but does not establish therapeutic efficacy or broad in vivo delivery performance.

Simplified

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy. Twin prime-editing guide RNAs have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs. Here we describe a prime assembly (PA) approach for the insertion of large DNA donor fragments, of which the ends are designed to overlap with the flaps generated by twin prime editing (twinPE). We used PA to insert one or multiple overlapping DNA fragments, with total insertion sizes ranging from 0.1 kb to 11 kb. An inhibitor of non-homologous end joining enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced, does not require co-delivery of exogenous DNA-dependent DNA polymerases and proceeds in non-cycling cells, suggesting independence from canonical homology-directed repair pathways. Our study demonstrates that PA can initiate Gibson-like assembly in cells to generate gene insertions without double-stranded DNA breaks, recombinases or homology-directed repair. 1,2 3-6

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Using prime assembly with linear DNA to insert large genetic sequences

Abstract

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