Targeted Transition Mutations in Phage-Assisted Evolution.

Phage-assisted evolution is a powerful strategy for directed evolution in which the protein undergoes continuous mutagenesis under selective pressure to rapidly evolve protein variants. However, conventional approaches often introduce mutations across the entire target plasmid, not just the gene-of-interest (GOI), leading to unwanted genomic changes that enable the evasion of selective pressure. To overcome this limitation, we integrated the MutaT7 system, which uses two base deaminases, into phage-assisted evolution to direct all four transition mutations specifically to the GOI. By using these deaminases, our system promotes the accumulation of all transition mutations, providing greater diversification of the GOI. This targeted yet comprehensive mutation strategy accelerates and enhances the evolutionary outcome of proteins to improve their stability, binding affinity, and specificity.