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

Autor: Zheng, Chunwei, Liu, Bin, Dong, Xiaolong, Gaston, Nicholas, Sontheimer, Erik J., Xue, Wen
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Zdroj: Nature Communications; 6/8/2023, Vol. 14 Issue 1, p1-9, 9p
Abstrakt: 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. Retrotransposons replicate their genetic information through target-primed reverse transcription (TPRT). Here the authors report a template-jumping prime editor (TJ-PE) to act similarly to TPRT and achieve insertions of large DNA fragments at endogenous sites: they rewrite a mutated exon in the mouse liver. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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