Development of a versatile Cas9-driven subpopulation-selection toolbox inLactococcus lactis.

5 years ago

Development of a versatile Cas9-driven subpopulation-selection toolbox inLactococcus lactis.

Peter A Bron, Jennelle K James, Simon van der Els, Michiel Kleerebezem

CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here we describe the extension of the Cas9 application toolbox to the industrially important dairy speciesLactococcus lactisThe Cas9 expression vector pLABTarget was constructed, encoding theStreptocccus pyogenesCas9 under control of a constitutive promoter, and allowing plug and play introduction of sgRNA sequences to target specific genetic loci. Introduction of apepN-targeting derivative of pLABTarget intoL. lactisMG1363 led to a strong reduction in the number of transformants obtained, which did not occur in apepNdeletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9 mediated double strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of apepNdeletion subpopulation from its corresponding single crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives inL. lactisFinally, pLABTarget containing sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages and integrative conjugative elements (ICE). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting inL. lactis, while the broad host range vector used suggests that this toolbox could be readily expanded to other Gram-positive bacteria.Significance statementMobile genetic elements inLactococcus lactisand other lactic acid bacteria play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 towards mobile genetic elements enabled their effective curing, which is of particular interest in the context of potentially problematic prophages present in a strain. Moreover, Cas9 targeting to other mobile genetic elements enables the deciphering of their contribution to dairy fermentation processes and further establishing their importance for product characteristics.

Publisher URL: http://doi.org/10.1128/AEM.02752-17

DOI: 10.1128/AEM.02752-17