Harnessing Diversity Generating Retroelements for in vivo targeted hyper-mutagenesis

Abstract

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 {lambda} GpJ receptor binding domain, and to its lamB receptor, DGRec created diverse variants enabling E. coli to evade infection, and {lambda} to reinfect lamB mutants.