Unlocking genetic potential: harnessing phage for targeted mutagenesis in phage-assisted evolution.

A critical challenge in DNA library genesis for evolution systems is avoiding off-target mutations, which can introduce undesirable changes elsewhere in the plasmid or even the host's genome: such mutations can allow cells or phages to propagate regardless of selective pressure. To construct a diverse pool of genes with mutations restricted to the gene-of-interest (GOI), we merged the strengths of phage-assisted evolution with the MutaT7 system for targeted mutagenesis. Hence, phage infection transfers genetic material to host cells where the MutaT7 system initiates targeted gene alteration. Our viral eMPAE system (enhanced mutation phage-assisted evolution) has three significant advantages over current state-of-the-art. (i) Up to 9 mutations in the GOI at a mutation rate of 5.6 mutations kb-1 day-1; in comparison, two of the most active mutagenesis plasmids, eMutaT7 and MP6, showed just one or no mutations in the GOI under phage-assisted conditions. (ii) No off-target mutations in the entire 6600 base-pair plasmid carrying the GOI; in contrast, MP6 mutations are completely untargeted. (iii) Our system allows easy substitution of GOI and DNA-modifying enzymes through unique restriction sites. Our proof-of-concept evolution experiment validates that protein variants obtained from eMPAE can withstand selective pressure and outperform the parent protein in binding assays. With its superior targeted mutagenesis ability, eMPAE is a transformative tool for constructing a diverse library of mutants. It is especially valuable for directed evolution, as its off-target mutation rate of <6.2 × 10-3 mutations kb-1 day-1 in the target plasmid minimizes the risk of creating cheater phages that circumvent selection.