Journal
NATURE COMMUNICATIONS
Volume 14, Issue 1, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41467-023-39137-6
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Targeted insertion of large DNA fragments using prime editing (PE) has shown promise for genome engineering and gene therapy. However, the efficiency of inserting larger fragments (>400bp) remains low and in vivo application has not been demonstrated. Inspired by retrotransposons, researchers have developed a template-jumping (TJ) PE approach which allows for the insertion of large DNA fragments with high efficiency and without double-stranded DNA breaks.
Targeted insertion of large DNA fragments holds promise for genome engineering and gene therapy. Prime editing (PE) effectively inserts short (<50bp) 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 (>400bp) 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 200bp and 500bp fragments with up to 50.5 and 11.4% efficiency, respectively, and enables GFP (similar to 800bp) 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.
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