Diversity-generating retroelements for programmable targeted hypermutagenesis

Diversity-generating retroelements (DGRs) are natural systems that accelerate the evolution of diverse bacterial functions through targeted hypermutation. We establish a method using DGRs coupled to recombineering (DGRec), which enables the diversification of any sequence of interest in Escherichiacoli. Detailed characterization of reverse transcriptase sequence biases demonstrates how it maximizes the exploration of the sequence space while avoiding nonsense mutations. By leveraging the high error rate of the DGR reverse transcriptase at adenines, DGRec can efficiently diversify user-defined sequence windows of 50–200 bp. Mutations can be focused at specific positions, with rates reaching up to 1.38 × 10⁻² per base per generation, allowing up to 24 mutations to accumulate within a single target sequence after 48 h. We apply DGRec to phage λ host-range engineering, to the evolution of dCas9 variants and to accelerated evolution of specific nanobodies through a bacterial display setup. Lastly, we establish the feasibility of DGR-mediated mutagenesis in yeast by adapting a recombination and selection strategy previously developed for retrons.