Chromosomal and Plasmid-Based CRISPRi Platforms for Conditional Gene Silencing in Lactococcus lactis
Year: 2025
Type: article
Abstract: Inducible CRISPR interference (CRISPRi) systems were established in Lactococcus lactis using both plasmid and chromosomal approaches. Expression of nuclease-deficient Cas9 (dCas9) from Streptococcus pyogenes was placed under the control of the nisin-inducible promoter PnisA, while sgRNAs were transcribed from the constitutive Pusp45 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 dCas9 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 ybeY, 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 ybeY-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 L. lactis, particularly for studying essential genes that cannot be deleted.
DOI: 10.3390/ijms26199516
Authors: Chenxi Huang, Meishan Liu, Jan Kok
Institutions: Wuhan University, University of Groningen
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,499
Funder: China Scholarship Council
Awards: G2692145841, G7619294028
OpenAlex ID: W4414653537