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.


Prime 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.

Keywords: AAV; mouse liver; prime editing; reverse transcriptase; split prime editor.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Gene Editing*
  • Liver
  • Mammals
  • Mice
  • Proprotein Convertase 9* / genetics
  • RNA-Directed DNA Polymerase
  • Ribonuclease H


  • RNA-Directed DNA Polymerase
  • Ribonuclease H
  • PCSK9 protein, human
  • Proprotein Convertase 9