Exploring the Evolutionary Landscape with Targeted In Vivo Hypermutations

Directed evolution has revolutionized protein engineering by applying the principles of natural selection to the laboratory. However, traditional in vitro methods are quite labor-intensive, while common in vivo methods suffer from low mutation rates and high rates of off-target mutations. To address these issues, researchers have developed targeted mutagenesis tools for rapid in vivo evolution of biomolecules. In this review, we discuss recent in vivo hypermutation tools that enable rapid sampling of the vast evolutionary landscape, all while supporting simultaneous selection of the best proteins within living organisms. We focus on three main mechanisms of hypermutation: (i) orthogonal replication, which uses error-prone replication machinery to replicate the target gene with low fidelity; (ii) CRISPR-Cas-guided mutators, where mutagenic proteins are localized to virtually any user-defined loci; and (iii) transcription-coupled mutagenesis, a simple, yet elegant tool that exploits the innate processivity of orthogonal ribonucleic acid (RNA) polymerases to guide mutagenic proteins along the target gene during transcription. We highlight key advantages of these systems, as well as some clinically- and biotechnology-relevant applications. We discuss important limitations and how they could be addressed in the future to make hypermutation tools with broad mutational spectra and windows that span entire genes with minimal off-target effects.