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Editing bacterial genomes is an essential tool in research and synthetic biology applications. Here, we describe multiplex genome editing by natural transformation (MuGENT), a method for accelerated evolution based on the cotransformation of unlinked genetic markers in naturally competent microorganisms. We found that natural cotransformation allows scarless genome editing at unprecedented frequencies of ∼50%. Using DNA substrates with randomized nucleotides, we found no evidence for bias during natural cotransformation, indicating that this method can be used for directed evolution studies. Furthermore, we found that natural cotransformation is an effective method for multiplex genome editing. Because MuGENT does not require selection at edited loci in cis, output mutant pools are highly complex, and strains may have any number and combination of the multiplexed genome edits. We demonstrate the utility of this technique in metabolic and phenotypic engineering by optimizing natural transformation in Vibrio cholerae. This was accomplished by combinatorially editing the genome via gene deletions and promoter replacements and by tuning translation initiation of five genes involved in the process of natural competence and transformation. MuGENT allowed for the generation of a complex mutant pool in 1 wk and resulted in the selection of a genetically edited strain with a 30-fold improvement in natural transformation. We also demonstrate the efficacy of this technique in Streptococcus pneumoniae and highlight the potential for MuGENT to be used in multiplex genetic interaction analysis. Thus, MuGENT is a broadly applicable platform for accelerated evolution and genetic interaction studies in diverse naturally competent species.
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