A truncated reverse transcriptase enhances prime editing by split AAV vectors

Prime editing is a new CRISPR-based, genome-editing technol-ogy 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 miti-gate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient deliv-ery 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 advan-tage (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 effi- ciency. We also show that this minimized PE gives rise to supe-rior 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% pre-cise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.

Automated summary

Prime editing is a novel genome editing technology that allows for more efficient and precise editing of the genome. This study made advancements in improving editing efficiencies by optimizing the reverse transcriptase moiety and addressing limitations of viral vector size. These improvements resulted in a codon-optimized and size-minimized prime editor that showed superior editing efficiency in in vivo applications.