Improved split prime editors enable efficient in vivo genome editing

Prime editor (PE) is a precise genome-editing tool capable of all possible base conversions, as well as insertions and deletions without DSBs or donor DNA. The efficient delivery of PE 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), editing efficiency at different gene loci varies among split sites, and efficient split sites within Cas9 nickase are limited. In this study, by screening multiple split sites, we demonstrated a series of efficient split site when delivering PE by dual-AAV. Additionally, we utilized a feature reported by others recently that RNase could be detached from the Cas9n and designed split sites in the first half of Cas9n. To test the editing efficiency in vivo, a novel dual-AAV split-ePE3 was packaged in AAV9 and delivered via tail vein injection in mice, achieving 24.4% precise genome editing 3 weeks post-injection. Our findings establish an alternative split-PE architecture that could achieve robust gene editing efficiency, facilitating the potential utility both in model organisms and as a therapeutic modality. Overall design: Split PE from different split sites was cloned into the AAV genome plasmid, and a series of split-PE systems were established by employing different promoters, truncated reverse transcriptase and untethered reverse transcriptase under the size restriction condition of the AAV packaging. Cells transfected with plasmids and mice infected with dual-AAV virus, the desired editing was integrated into the genome of host cells. Cells were harvested 72 hours after plasmid transfection, and mouse livers were collected 3 weeks after the virus injection into the tail vein. To detect the outcome of prime editing, we amplified the gDNA harvested from the edited cells and subjected the amplicons to high-throughput sequencing. Additional data for this study are in: GSE291960