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Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis.

Simon van der Els1,2,3, Jennelle K James2, Michiel Kleerebezem4,3

  • 1Host-Microbe Interactomics Group, Animal Sciences, Wageningen University & Research, Wageningen, The Netherlands.

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|February 18, 2018
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Summary
This summary is machine-generated.

We developed the pLABTarget CRISPR-Cas9 tool for genetic engineering in Lactococcus lactis. This system efficiently targets and removes mobile genetic elements and aids in gene deletion, improving dairy fermentation research.

Keywords:
CRISPR-Cas9Lactococcus lactisgenetic engineering

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetic Engineering

Background:

  • CRISPR-Cas9 technology is a powerful tool for genetic modification in various organisms.
  • Lactococcus lactis is a key bacterium in dairy fermentation, with mobile genetic elements influencing its properties.
  • Existing genetic tools for L. lactis have limitations in efficiency and scope.

Purpose of the Study:

  • To adapt and validate the CRISPR-Cas9 system for genetic manipulation in Lactococcus lactis.
  • To develop a versatile vector (pLABTarget) for targeting specific genetic loci and mobile genetic elements.
  • To demonstrate the utility of this system for gene deletion and mobile genetic element removal in L. lactis.

Main Methods:

  • Construction of the Cas9 expression vector pLABTarget, enabling customizable short guide RNA (sgRNA) integration.
  • Introduction of pLABTarget derivatives into L. lactis strains to target the pepN gene and mobile genetic elements.
  • Assessment of Cas9-mediated lethality, gene deletion efficiency, and mobile genetic element curing.

Main Results:

  • Cas9-mediated double-strand breaks were shown to be specific and lethal in the L. lactis chromosome.
  • The pLABTarget system facilitated the selection of pepN deletion mutants, accelerating genetic engineering.
  • Effective curing of plasmids, prophages, and integrative conjugative elements (ICEs) was achieved using pLABTarget.

Conclusions:

  • The pLABTarget vector provides an efficient CRISPR-Cas9-based toolbox for genetic engineering in L. lactis.
  • This technology enables precise gene deletion and the removal of mobile genetic elements, crucial for understanding dairy fermentation.
  • The broad-host-range nature of the vector suggests potential applications in other Gram-positive bacteria.