Harnessing Diversity Generating Retroelements for in vivo targeted hyper-mutagenesis

The rapid evolution of novel functions requires targeted mutagenesis to avoid harmful mutations. Diversity-generating retroelements (DGRs) are natural systems that accelerate the evolution of diverse bacterial functions through targeted hypermutation. Here, we establish a method utilizing DGRs coupled to recombineering (DGRec), enabling the diversification of any sequence of interest in E. coli . DGRec can programmably diversify specific residues by leveraging the high error rate of the DGR reverse-transcriptase at adenines. We perform a detailed characterization of the reverse-transcriptase biases, highlighting how it maximizes the exploration of the sequence space while avoiding nonsense mutations. Applied to the phage λ GpJ receptor binding domain, and to its lamB receptor, DGRec created diverse variants enabling E. coli to evade infection, and λ to reinfect lamB mutants.