Template-jumping prime editing enables large insertion and exon rewriting in vivo.

Targeted insertion of large DNA fragments holds promise for genome engineering and gene therapy. Prime editing (PE) effectively inserts short (<50 bp) sequences. Employing paired prime editing guide RNAs (pegRNAs) has enabled PE to better mediate relatively large insertions in vitro, but the efficiency of larger insertions (>400 bp) remains low and in vivo application has not been demonstrated. Inspired by the efficient genomic insertion mechanism of retrotransposons, we develop a template-jumping (TJ) PE approach for the insertion of large DNA fragments using a single pegRNA. TJ-pegRNA harbors the insertion sequence as well as two primer binding sites (PBSs), with one PBS matching a nicking sgRNA site. TJ-PE precisely inserts 200 bp and 500 bp fragments with up to 50.5 and 11.4% efficiency, respectively, and enables GFP (~800 bp) insertion and expression in cells. We transcribe split circular TJ-petRNA in vitro via a permuted group I catalytic intron for non-viral delivery in cells. Finally, we demonstrate that TJ-PE can rewrite an exon in the liver of tyrosinemia I mice to reverse the disease phenotype. TJ-PE has the potential to insert large DNA fragments without double-stranded DNA breaks and facilitate mutation hotspot exon rewriting in vivo.