Chromosomal and Plasmid-Based CRISPRi Platforms for Conditional Gene Silencing in Lactococcus lactis

Area of Science:

• Microbiology
• Molecular Biology
• Genomics

Background:

• CRISPR interference (CRISPRi) is a powerful tool for gene regulation.
• Developing inducible systems in Lactococcus lactis is crucial for functional genomics.

Purpose of the Study:

• To establish and compare plasmid-based and chromosome-based inducible CRISPRi systems in Lactococcus lactis.
• To assess the efficiency and regulation of these systems for gene silencing.

Main Methods:

• Constructed inducible CRISPRi systems using nuclease-deficient Cas9 (dCas9) and sgRNAs.
• Utilized nisin-inducible (PnisA) and constitutive (Pusp45) promoters.
• Integrated dCas9 into the chromosome for improved regulation.
• Performed gene silencing of reporter genes, AcmA, and essential gene ybeY.
• Conducted transcriptome analysis of ybeY-silenced cells.

Main Results:

• Both plasmid and chromosomal systems achieved gene repression.
• Plasmid systems exhibited leaky expression, while chromosomal integration ensured tight regulation.
• Chromosome-based CRISPRi (cbCRISPRi) successfully silenced the essential gene ybeY, causing growth defects.
• Transcriptome analysis revealed significant impacts on ribosomal proteins, membrane proteins, and transporters.

Conclusions:

• Inducible CRISPRi systems, particularly the chromosome-based platform, provide robust tools for gene function studies in L. lactis.
• These systems enable the investigation of essential genes that are otherwise difficult to study.
• The cbCRISPRi system offers precise control for functional genomics research in L. lactis.