SLICER: Seamless Loss of Integrated Cassettes Using Endonuclease Cleavage and Recombination inDeinococcus radiodurans

Abstract

ABSTRACTMethods for creating seamless genome modifications are an essential part of the microbial genetic toolkit that allows for strain engineering through the recycling of selectable markers. Here, we report the development of a method, termed SLICER, which can be used to create seamless genome modifications inD. radiodurans. We used SLICER to sequentially target four putative restriction-modification (R-M) system genes, recycling the same selective and screening markers for each subsequent deletion. A fifth R-M gene was replaced by a selectable marker to create a finalD. radioduransstrain with 5 of the 6 putative R-M systems deleted. While we observed no significant increase in transformation efficiency, SLICER is a promising method to obtain a fully restriction-minus strain and expand the synthetic biology applications ofD. radioduransincluding as anin vivoDNA assembly platform.GRAPHICAL ABSTRACT