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A truncated reverse transcriptase enhances prime editing by split AAV vectors

A truncated reverse transcriptase enhances prime editing by split AAV vectors

Authors :
Zongliang Gao
Sujan Ravendran
Nanna S. Mikkelsen
Jakob Haldrup
Huiqiang Cai
Xiangning Ding
Søren R. Paludan
Martin K. Thomsen
Jacob Giehm Mikkelsen
Rasmus O. Bak
Source :
Gao, Z, Ravendran, S, Mikkelsen, N S, Haldrup, J, Cai, H, Ding, X, Paludan, S R, Thomsen, M K, Mikkelsen, J G & Bak, R O 2022, ' A truncated reverse transcriptase enhances prime editing by split AAV vectors ', Molecular Therapy, vol. 30, no. 9, pp. 2942-2951 . https://doi.org/10.1016/j.ymthe.2022.07.001
Publication Year :
2022
Publisher :
Elsevier BV, 2022.

Abstract

Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.

Details

ISSN :
15250016
Volume :
30
Database :
OpenAIRE
Journal :
Molecular Therapy
Accession number :
edsair.doi.dedup.....e482d3b5e2fcbc3c0e1a39faf07598db
Full Text :
https://doi.org/10.1016/j.ymthe.2022.07.001