Efficient PE system PE7-scFv-MLH1dn by optimizing the configuration of La and MLH1dn.

Prime editing is a versatile gene editing technology that has undergone significant enhancements since its inception. Currently, a recently reported prime editing system, PE7 combined with MLH1dn, incorporates two auxiliary components designed to enhance editing efficiency: La fused to the reverse transcriptase (RT) and non-recruited MLH1dn. We hypothesized that the expression and recruitment strategies for these proteins could be further optimized. In this study, we first employed the SunTag system to recruit La and MLH1dn, either individually or simultaneously, and found that recruitment, particularly co-recruitment, significantly enhanced prime editing efficiency. Subsequently, we evaluated various expression and recruitment configurations and determined that a PE system utilizing 2∗GCN4-anchored scFv-fused MLH1dn recruitment, while retaining La fused to RT, achieved the highest editing efficiency. Testing across multiple genomic loci demonstrated that this auxiliary factor-based strategy further improved editing efficiency compared with PE7 alone. In summary, by systematically exploring the recruitment modalities of auxiliary factors, we have developed a more efficient prime editing system, providing researchers with a state-of-the-art tool for high-efficiency genome modification.