Development of a flexible split prime editor using truncated reverse transcriptase

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, the potential effect of overexpressing RT in mammalian cells is largely unknown. Here, we developed a compact PE with complete deletion of the RNase H domain of reverse transcriptase (RT), which showed comparable editing to full-length PE. Using compact PE, we tested the effect of 4 different Cas9 split sites and found that the Glu 573 split site supports robust editing (up to 93% of full-length PE). The compact PE, but not PE2, abolished its binding to eRF1 and showed minimal effect on stop codon readthrough, which therefore might reduce the effects on protein biosynthesis. This study identifies a safe and efficient compact PE2 that enables flexible split-PE design to facilitate efficient delivery in vivo and advance the utility of prime editing.