Prime assembly with linear DNA donors enables large genomic insertions

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