Abstract
The generation of a library of variant genes is a prerequisite of directed evolution, a powerful tool for biomolecular engineering. As the number of all possible sequences often far exceeds the diversity of a practical library, methods that allow efficient library diversification in living cells are essential for in vivo directed evolution technologies to effectively sample the sequence space and allow hits to emerge. While traditional whole-genome mutagenesis often results in toxicity and the emergence of "cheater" mutations, recent developments that exploit the targeting and editing abilities of genome editors to facilitate in vivo library diversification have allowed for precise mutagenesis focused on specific genes of interest, higher mutational density, and reduced the occurrence of cheater mutations. This minireview summarizes recent advances in genome editor-directed in vivo library diversification and provides an outlook on their future applications in chemical biology.