Versatile Cas9-driven subpopulation selection toolbox for Lactococcus lactis

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

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

20 Citations (Scopus)


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 species Lactococcus lactis. The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN-targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion 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 a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis. Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis, while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria.
Original languageEnglish
Article numbere02752-17
JournalApplied and Environmental Microbiology
Issue number8
Publication statusPublished - 1 Apr 2018


  • CRISPR-Cas9
  • Genetic engineering
  • Lactococcus lactis

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