CRISPR-Cas9-mediated genome editing in vancomycin-resistant Enterococcus faecium.

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Enterococcus faecium is a growing cause of hospital-acquired infections.
• Antibiotic resistance in E. faecium poses a significant public health threat.
• Generating targeted mutants in E. faecium is crucial for research but is currently slow and labor-intensive.

Purpose of the Study:

• To develop a more efficient method for generating targeted genome modifications in E. faecium.
• To adapt the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 system for use in E. faecium.

Main Methods:

• Utilized the high recombination rates of E. faecium.
• Applied the CRISPR-Cas9 genome editing tool.
• Generated a lacL deletion mutant and inserted green fluorescent protein (GFP) into the msrC gene.

Main Results:

• Successfully generated targeted genome modifications in E. faecium.
• Reduced the time required for mutant generation from 4-5 weeks to 3 weeks.
• Demonstrated the ability to create both gene deletions and insertions using CRISPR-Cas9.
• Distinguished wild-type from mutant strains using blue/white screening with X-gal.
• Created stable green fluorescent E. faecium cells by inserting GFP.

Conclusions:

• CRISPR-Cas9 is an efficient tool for generating targeted genome modifications in E. faecium.
• The developed method significantly reduces the time and labor required for mutant generation.
• This approach has the potential for application in other Gram-positive bacteria with high recombination rates.