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A rapid and versatile tool for genomic engineering in Lactococcus lactis.

Tingting Guo1, Yongping Xin1, Yi Zhang1

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Researchers developed a new genome editing tool for Lactococcus lactis using ssDNA recombineering and CRISPR/Cas9 counterselection. This precise and efficient method enables rapid genetic modifications for improved cell factory applications.

Keywords:
CRISPR/Cas9 counterselectionGenomic engineeringLactococcus lactisssDNA recombineering

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Area of Science:

  • Microbiology
  • Synthetic Biology
  • Molecular Biology

Background:

  • Lactococcus lactis is a well-characterized lactic acid bacterium extensively used in industrial applications.
  • It has been engineered into cell factories for producing valuable bioactive compounds.
  • There is a need for precise and efficient genome editing tools to enhance its engineered functionality.

Purpose of the Study:

  • To develop and optimize a novel genome engineering tool for Lactococcus lactis.
  • To combine single-stranded DNA (ssDNA) recombineering with CRISPR/Cas9 counterselection for precise genetic modifications.
  • To enhance the efficiency and versatility of genome editing in L. lactis.

Main Methods:

  • Selection of a highly active RecT recombinase from Enterococcus faecalis for ssDNA homologous recombination.
  • Application of the CRISPR/Cas9 system for counterselection to screen mutants lacking a selectable phenotype.
  • Optimization of CRISPR/Cas9 parameters (plasmid copy number, spacer length) to eliminate off-target mutations.
  • Sequential point mutations and seamless genomic deletions/insertions using the developed tool.

Main Results:

  • RecT recombinase mediated 100% efficient homologous recombination with ssDNA at the rpoB locus.
  • CRISPR/Cas9 counterselection achieved 46% efficiency for generating an upp mutant.
  • Optimized CRISPR/Cas9 system eliminated off-target mutations in recA, galK, hemN, and noxD genes.
  • Sequential point mutations in upp and galK achieved >75% efficiency.
  • Seamless genomic deletions (50/100 bp) and insertions (34 bp loxP site) were accomplished within 72 hours.

Conclusions:

  • A rapid, versatile, and precise genome engineering tool for L. lactis was developed.
  • The tool combines ssDNA recombineering with optimized CRISPR/Cas9 counterselection.
  • This method simplifies the production of isogenic strains for gene function assessment and biosynthetic host construction.