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

Inducible CRISPR interference (CRISPRi) systems were established in <i>Lactococcus lactis</i> using both plasmid and chromosomal approaches. Expression of nuclease-deficient Cas9 (dCas9) from <i>Streptococcus pyogenes</i> was placed under the control of the nisin-inducible promoter P_nisA, while sgRNAs were transcribed from the constitutive P_usp45 promoter. To monitor expression, dCas9 was fused with superfolder GFP. Plasmid-based constructs successfully repressed a luciferase reporter gene and silenced the gene of the major autolysin, AcmA, leading to the expected morphological phenotype. However, plasmid systems showed leaky expression, producing mutant phenotypes even without induction. Chromosomal integration of <i>dCas9</i> reduced its expression level by approximately 20-fold compared with plasmid-based expression, thereby preventing leaky activity and ensuring tight regulation. This chromosome-based (cbCRISPRi) platform enabled controlled repression of the essential gene <i>ybeY</i>, which resulted in severe growth defects. Restoration of wild-type phenotypes was achieved by introducing a synonymous codon substitution in the sgRNA target region. Transcriptome analysis of <i>ybeY</i>-silenced cells revealed downregulation of ribosomal protein genes and widespread effects on membrane-associated proteins, ATP synthase subunits, and various transporters. These inducible CRISPRi platforms provide robust and tunable tools for functional genomics in <i>L. lactis</i>, particularly for studying essential genes that cannot be deleted.