A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver
UMass Chan AffiliationsLi Weibo Institute for Rare Diseases Research
Molecular, Cell and Cancer Biology
Program in Molecular Medicine
RNA Therapeutics Institute
Document TypeJournal Article
MetadataShow full item record
AbstractPrime editor (PE) has tremendous promise for gene therapy. However, it remains a challenge to deliver PE (>6.3 kb) in vivo. Although PE can be split into two fragments and delivered using dual adeno-associated viruses (AAVs), choice of split sites within Cas9-which affects editing efficiency-is limited due to the large size of PE. Furthermore, overexpressing reverse transcriptase in mammalian cells might disrupt translation termination via its RNase H domain. Here, we developed a compact PE without the RNase H domain that showed editing comparable with full-length PE. With compact PE, we used a Cas9 split site (Glu 573) that supported robust editing in cells (up to 93% of full-length PE) and in mouse liver. We then demonstrated that split-cPE573 delivered by dual-AAV8 efficiently mediated a 3-bp TGA insertion in the Pcsk9 gene in mouse liver. Compact PE without the RNase H domain abolished its binding to peptidyl release factor 1 (eRF1) and mitigated the stop codon readthrough effect observed with full-length PE. This study identifies a compact PE with a flexible split design to advance utility of prime editing in vivo.
SourceZheng C, Liang SQ, Liu B, Liu P, Kwan SY, Wolfe SA, Xue W. A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver. Mol Ther. 2022 Mar 2;30(3):1343-1351. doi: 10.1016/j.ymthe.2022.01.005. Epub 2022 Jan 5. PMID: 34998953; PMCID: PMC8899602.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/52193
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Except where otherwise noted, this item's license is described as Copyright © 2022 The American Society of Gene and Cell Therapy. Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0); Attribution-NonCommercial-NoDerivatives 4.0 International