Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis

Abstract

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. IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) 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 toward 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 of other mobile genetic elements enables the deciphering of their contribution to dairy fermentation processes and further establishment of their importance for product characteristics.

An open microscopy framework suited for tracking dCas9 in live bacteria

“…advent of super-resolution microscopy has been providing additional means to gain insights in biology 1 . In 33 addition to sub-diffractive imaging of cellular structures, super-resolution microscopy can be used to 34 observe the dynamics and interactions of individual proteins in living cells via single particle tracking photo-35 activatable localization microscopy (sptPALM) [2][3][4] . Compared to imaging applications, in vivo sptPALM has 36 additional experimental challenges to overcome: increased cellular background fluorescence, limited 37 photon budget of single genetically encoded fluorescent proteins and a high time resolution (millisecond 38 range) required to obtain molecular tracks.…”

Safety Aspects of Genetically Modified Lactic Acid Bacteria

“…Its application to DNA targeting of four genomic islands in Streptococcus thermophilus resulted in generation of stable mutants that collectively lacked a total of 7% of their genome [45]. Cas9 has also been used for removal of plasmids, integrative conjugative elements, and prophages in L. lactis, where specific genetic loci were targeted using the pNZCRISPR and pLABTarget vectors [46,47].…”

Renaissance of traditional DNA transfer strategies for improvement of industrial lactic acid bacteria

“…These conjugative MGEs encode similar type IV secretion mobilization machineries that are involved in oriT-dependent conjugal transfer to appropriate recipient cells, but also encode distinct functions involved in chromosomal integration and excision (ICEs), and extra-chromosomal replication (plasmids) [14 ,15]. The genetically conserved functions of these conjugative MGEs have been exploited in tools aiming to detect them in bacterial genome sequences [16 ,17,18], while delimitation of ICEs can be achieved by pan-genome and core-genome mapping [16 ] or by curing them from the host chromosome [19 ]. Besides their canonical functions, the conjugative MGEs encode a variable number of accessory genes ('cargo') that confer phenotypes to host cells [15,20].…”