Single-plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Lactococcus lactis is a crucial food-grade bacterium widely utilized in food and medical sectors.
• Traditional gene knockout in L. lactis relies on RecA-dependent homologous recombination, which is inefficient and labor-intensive.
• Engineering L. lactis is valuable for producing recombinant proteins due to its simple metabolism and small genome.

Purpose of the Study:

• To develop a precise and efficient genome-editing plasmid for L. lactis NZ9000.
• To optimize gene deletion efficiency using CRISPR-Cas9 technology.
• To establish a novel tool for advancing L. lactis genome engineering.

Main Methods:

• Development of a CRISPR-Cas9 based genome-editing plasmid, designated pLL.
• Optimization of gene deletion efficiency by studying various single guide RNA (sgRNA) promoters.
• Demonstration of sequential gene deletion and gene identification using the pLL system.

Main Results:

• Achieved up to 50% gene deletion efficiency for LLNZ_02045 (ldh).
• Successfully performed sequential gene deletion of LLNZ_11240 (upp) and LLNZ_04580 (upp1).
• Identified the gene encoding uracil phosphoribosyltransferase using the developed system.
• Confirmed that sgRNA binding location does not impact gene deletion efficiency.

Conclusions:

• The pLL plasmid provides a robust and efficient tool for L. lactis NZ9000 genome engineering.
• This novel system surpasses traditional methods in speed and precision for gene manipulation.
• The developed tool facilitates further research and understanding of the L. lactis NZ9000 genome.