Abstract

 

Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants with the desired properties. However, DE methods have so far been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools are being developed to facilitate DE in native genetic environments of multicellular organisms that circumvent enduring barriers like longer life cycle, small library sizes, and low mutation rates. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE systems by enabling base substitutions and randomization of targeted DNA sequences. BE-based TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements.