Prime assembly with linear DNA donors enables large genomic insertions

Apr 29, 2026Nature

Using prime assembly with linear DNA to insert large genetic sequences

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Abstract

Insertion sizes of DNA fragments ranged from 0.1 kb to 11 kb using a prime assembly approach.

The prime assembly approach enables the insertion of large DNA fragments that overlap with flaps generated by twin prime editing.
Efficiency and precision of DNA insertions were enhanced by using an inhibitor of non-homologous end joining.
This method can operate in non-cycling cells and does not require the delivery of external DNA polymerases.
Prime assembly may function independently of traditional DNA repair pathways.

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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