Development of a flexible split prime editor using truncated reverse transcriptase

Abstract

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.

New CRISPR Tools to Correct Pathogenic Mutations in Usher Syndrome

“…Zheng et al also removed the RNase H domain, using the truncated construct to mediate codon insertion into the Pcsk9 gene in mouse liver cells [ 59 ]. Further truncations into the connection domain also lead to a reverse transcriptase that retained a high percentage of activity [ 60 ]. The first 23 amino acids are not essential to the M-MLV function [ 61 ].…”

Precision genome editing in plants using gene targeting and prime editing: existing and emerging strategies

“…Thus, the efficacy of PE in plants may be improved using transformation systems that deliver large quantities of the PE construct (e.g., the geminivirus system). Alternatively, the components of PE constructs (e.g., Cas9) can be split into multiple parts [112][113][114] and delivered to the plant using nanoparticles [115][116][117] or virus-based transformation. [114] In this example, the nCas9-RT protein could be reconstituted in the cells using an intein sequence that would enable the various parts of the fusion protein to be rejoined after trans-splicing.…”