A truncated reverse transcriptase enhances prime editing by split AAV vectors

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*

*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review


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.

Original languageEnglish
JournalMolecular Therapy
Pages (from-to)2942-2951
Number of pages10
Publication statusPublished - Sept 2022


  • AAV vectors
  • CRISPR-Cas9
  • gene editing
  • gene therapy
  • in vivo delivery
  • prime editing
  • reverse transcriptase
  • Gene Editing
  • Dependovirus/genetics
  • Animals
  • CRISPR-Cas Systems
  • Genetic Vectors/genetics
  • Proprotein Convertase 9/genetics
  • Mice
  • RNA, Guide, Kinetoplastida/genetics
  • RNA-Directed DNA Polymerase/genetics


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