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

## TL;DR

Researchers developed inducible CRISPRi systems in Lactococcus lactis using plasmid and chromosomal approaches to control gene silencing, with the chromosomal method offering tighter regulation.

## Contribution

A novel chromosomal CRISPRi platform was developed to enable precise and leak-free gene silencing in Lactococcus lactis.

## Key findings

- Plasmid-based CRISPRi systems showed leaky expression, causing unintended gene silencing.
- Chromosomal integration of dCas9 reduced expression by 20-fold and prevented leaky activity.
- Silencing of ybeY caused growth defects, and transcriptome analysis revealed widespread gene downregulation.

## 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.