Improved split prime editors enable efficient in vivo genome editing

Efficient prime editor (PE) delivery in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), the editing efficiency at different gene loci varies among split sites. Furthermore, efficient split sites within Cas9 nickase (Cas9n) are limited. Here, we verified that 1115 (Asn) is an efficient split site when delivering PEs by dual AAVs. Additionally, we utilized a feature in which reverse transcriptase could be detached from the Cas9n and designed split sites in the first half of Cas9n. We found that split-PE-367 enabled high editing efficiency with Rma intein. To test the editing efficiency in vivo, split-ePE3-367 was packaged in AAV9 and achieved 17.5% precise editing in mice. Our findings establish an alternative split-PE architecture that enables robust editing efficiency, facilitating potential utility in disease modeling and correction.