Easy-Curing and pH-Regulated CRISPR-Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris

Area of Science:

• Microbiology
• Molecular Biology
• Genetic Engineering

Background:

• Lactococcus cremoris is a key dairy starter culture, but genetic manipulation can be challenging.
• Existing methods for genetic modification in L. cremoris often require selective pressure and can be inefficient.

Purpose of the Study:

• To develop a versatile and efficient CRISPR-Cas9 based system for genetic editing in Lactococcus cremoris.
• To enable both plasmid and chromosome editing, including the generation of plasmid-free strains.

Main Methods:

• Construction of shuttle vectors expressing Cas9 nuclease and single-guide RNA under inducible and constitutive promoters.
• Utilized a pH-regulated promoter for tunable Cas9 expression and a strong constitutive promoter for sgRNA.
• Implemented a host-independent recombination system (λRed-phage Redβ recombinase and EcSSB) for enhanced chromosome editing.

Main Results:

• Demonstrated efficient plasmid curing of low- and high-copy number plasmids in L. cremoris.
• Achieved tunable targeting efficiency by regulating Cas9 expression.
• Successfully generated thousands of gene-edited cells using CRISPR-Cas9 coupled with endogenous or Redβ-EcSSB-assisted recombination.
• Observed no Cas9-associated toxicity and a high vector loss rate in the absence of selective pressure.

Conclusions:

• The developed CRISPR-Cas9 system provides a powerful and flexible tool for genetic engineering of L. cremoris.
• This strategy simplifies the generation of precisely modified, plasmid-free L. cremoris strains.
• The system's efficiency, tunability, and lack of toxicity make it highly valuable for dairy starter culture development.