Multiplex genome editing by natural transformation

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Abstract
Significance: The ability to generate mutants is essential in microbiology research. Although methods have been developed for making defined single mutations in bacterial genomes, methods for simultaneously generating multiple defined mutations (i.e., multiplex genome editing) have been limited to model species such asEscherichia coli. Diverse microbial species have the ability to take up exogenous DNA naturally and integrate it into their genome, a process known as “natural transformation.” Although natural transformation has been exploited for making single mutations, it has not been used previously for multiplex genome editing. Here, we describe multiplex genome editing by natural transformation and demonstrate the utility of this method in two naturally competent microbial species. This method should be broadly applicable for diverse research applications.

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