Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined stretches of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein segments that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA stretches of less than 30 amino acids, and these DNA stretches can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. The combination of MORPHING with classical directed evolution and semi-rational approaches, or with neutral genetic drift, may lead to the development of new adaptive pathways to engineer more robust biocatalysts.

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