Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced Sp Cas9 prime editor (PE) lacking the RNaseH domain (PE2 ^{Δ RnH} ) and an intein-split construct (PE2 p.1153) for adeno-associated virus–mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2 ^{Δ RnH} via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pah ^enu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10 ¹⁴ vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.

Prime editing is a versatile genome editing technology that circumvents the need for DNA double-strand break formation and homology-directed repair, making it particularly suitable for in vivo correction of pathogenic mutations. Here we developed liver-specific prime editing approaches with temporally restricted prime editor (PE) expression. We first established a dual-delivery approach where the prime editor guide RNA is continuously expressed from adeno-associated viral vectors and only the PE is transiently delivered as nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP). This strategy achieved 26.2% editing with PEmax and 47.4% editing with PE7 at the Dnmt1 locus using a single 2 mg kg⁻¹ dose of mRNA–LNP. When targeting the pathogenic Pah ^enu2 mutation in a phenylketonuria mouse model, gene correction rates reached 4.3% with PEmax and 20.7% with PE7 after three doses of 2 mg kg⁻¹ mRNA–LNP, effectively reducing blood l-phenylalanine levels from over 1,500 µmol l⁻¹ to below the therapeutic threshold of 360 µmol l⁻¹. Encouraged by the high efficiency of PE7, we next explored a simplified approach where PE7 mRNA was co-delivered with synthetic prime editor guide RNAs encapsulated in LNP. This strategy yielded 35.9% editing after two doses of RNA–LNP at the Dnmt1 locus and 8.0% editing after three doses of RNA–LNP at the Pah ^enu2 locus, again reducing l-phenylalanine levels below 360 µmol l⁻¹. These findings highlight the therapeutic potential of mRNA–LNP-based prime editing for treating phenylketonuria and other genetic liver diseases, offering a scalable and efficient platform for future clinical translation.