A Targeted Phage System for Directed Evolution

Directed evolution consists in the application of reiterative rounds of sequence variation and selection in the lab. This allows the engineering of genes and enzymes without rational cues. This is useful for poorly studied target genes or organisms. However, current directed evolution approaches rely mostly on global mutagenesis, which can reduce variant library sizes and hinder the selection of improved variants by introducing deleterious mutations outside of the target gene. On the other hand, targeted systems fail to cover large spans of DNA and lack a complete mutational spectrum ().

Current selection strategies often rely on manual screening of variants by the researcher, which is time consuming and subjective. We are developing a targeted sequence diversification system based on the phage T7 RNA polymerase and mutagenic enzymes, including activation-induced deaminase (AID) and error-prone DNA polymerase IV. Mutagenesis of targets is achieved during T7-dependent transcription. The on-target and off-target activity of the mutator will be analysed by reversion assays of mutated GFP and antibiotic resistance genes, for both genome-integrated and plasmid encoded targets. Subsequently, next generation sequencing will be employed to determine mutagenesis rates and spectrum.

Sequence variation will be followed by selection based on conditional M13 phage replication, which links target to functionality to phage fitness and can undergo several rounds of selection per day. A genetic network will be designed so that target gene variants that show improved activity up-regulate the expression of a phage infectivity gene (gVI), allowing infection of host cells and the increase in population frequency of beneficial variants. The phage-assisted evolution technique will be upgraded to include modules to regulate the stringency of selection and counter-selection, allowing the evolution of more complex targets. The final directed evolution platform will be put to test by attempting to engineer a set of orthogonal CI transcription factors. If successful, the technique will be optimised for other targets.