Westlake Genetech Advances Split Prime Editors for Efficient In Vivo Genome Editing

Recently,Westlake Genetech R&D TeamInCell ReportsPublished online titled“Improved split prime editors enable efficient in vivo genome editing”The paper.

Paper link: https://doi.org/10.1016/j.celrep.2024.115144

This study screened for efficient Split-PE cleavage sites (Fig. 1A), designed a new PE architecture, and achieved efficient in vivo gene editing by delivering PE via AAV.

Specifically, the Westlake Genetech research team tested multiple Split-PE cleavage sites in vitro and found that split-PE-1115 exhibited high editing efficiency (Fig. 1B-C). In addition, by using free reverse transcriptase (RT) and screening cleavage sites near the N-terminus of Cas9 nickase (Cas9n), they identified a highly efficient split-PE (split-PE-367) (Fig. 1B, D). Further comparisons with several existing split-PEs revealed that split-PE-1115 and split-PE-367 demonstrated superior PE editing efficiency across multiple genomic loci.

Figure 1 Efficient split-PE screening

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Finally, split-ePE3-367 was packaged into AAV9 for targeting the PCSK9 gene (a gene that regulates cholesterol homeostasis) in mice (Fig. 2A). The results showed that split-ePE3-367 achieved precise editing of mouse PCSK9 with a maximum efficiency of 17.5%, surpassing the best-reported strategy (12.9%) (Fig. 2B-C). Meanwhile, no significant editing was detected at potential off-target sites, demonstrating excellent safety.

Figure 2 Dual AAV-mediated split-ePE3-367 induces PCSK9 gene mutations in mice

In summary, this study expanded the screening window for separation sites and identified an efficient separation site (split-PE-367) by using free RT. By combining the optimized split-ePE3-367 with AAV, successful highly efficient and specific editing of target genes in mice was achieved, demonstrating the potential application of the dual AAV system delivering split-ePE3-367 in living models and its potential value for the treatment of genetic diseases.

In the future, the team will explore more effective separation sites and evaluate the efficiency and accuracy of novel PE systems for gene editing related to different diseases.