biostudies-literatureGao ZIndependent Research Fund DenmarkNovo Nordisk FoundationCarlsberg FoundationInnovationsfondenLundbeck FoundationNovo Nordisk Fonden2942-2951https://www.ebi.ac.uk/biostudies/studies/S-EPMC9481986biostudies-literatureUnknown30(9)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 advantaMolecular therapy : the journal of the American Society of Gene TherapyA truncated reverse transcriptase enhances prime editing by split AAV vectors.PMC9481986324-2019-1832NNF19OC0058238R303-2018-3571Haldrup JDing XRavendran SMikkelsen NSBak ROGao ZThomsen MKMikkelsen JGCai HPaludan SRfalseA truncated reverse transcriptase enhances prime editing by split AAV vectors.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 advanta2022-01-01T00:00:00Z2022 Sep2025-04-05T00:09:27.207Z2025-04-05T00:09:27.207ZS-EPMC94819863580882410.1016/j.ymthe.2022.07.001