Prime editing (PE) is a precise genome editing method that enables introduction of short insertions, deletions or substitutions without inducing double-strand breaks. It uses a fusion protein, consisting of a Cas9 nickase, which allows induction of a nick in a single DNA strand at the target site, and a reverse transcriptase, which catalyses reverse transcription from an RNA template encoding the novel sequence. PE has a great potential in therapeutic use, but in vivo delivery remains challenging due to the size of PE machinery, which exceeds the packaging capacities of some vectors. At the National Institute of Chemistry an improved version of the system called coiled-coil prime editing (ccPE) was developed, in which the two components are not covalently linked, but instead interact non-covalently via a coiled-coil (CC) motif interaction. The latter is formed between two CC-peptides that are covalently added to each subunit. This approach allows the two subunits to associate non-covalently into a functional complex, while keeping the system divided into two separate parts. This overcomes the size limitation by allowing each component to be delivered separately in its own vector. We compared the efficiency of ccPE with standard PE on different HEK293 cell endogenous loci and on a clinically relevant CTNNB1 gene mutation. We also tested ccPE with various CC-peptides. We found that ccPE performs equally or more effectively than PE. Additionally, we aimed to evaluate the efficacy of ccPE system in patient-derived induced pluripotent stem cells (iPSC) with a mutation at one allele of the CTNNB1 gene. We tested different delivery methods for introducing nucleic acids into iPSCs and of all methods tested, only lentiviral transduction proved effective, which is why we constructed plasmids for the preparations of lentiviral vectors encoding the PE and ccPE systems.
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