Short communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Lactococcus lactis is a crucial probiotic lactic acid bacterium (LAB) widely used in the dairy industry.
• Existing genetic modification methods for L. lactis are limited, hindering precise gene expression control.
• The nisin-controlled inducible expression system is the only commercial system available for LAB, but lacks robust gene repression tools.

Purpose of the Study:

• To develop a novel, inducible CRISPR-dCas9 system for gene transcription repression in L. lactis.
• To establish a versatile tool for modulating gene expression in L. lactis NZ9000.
• To identify novel functional genes, such as those involved in bile salt tolerance, using the developed system.

Main Methods:

• A two-plasmid system was constructed using inducible Pnisin and constitutive P44 promoters.
• Streptococcus pyogenes dCas9 and single-guide RNA (sgRNA) were expressed to target specific genes.
• CRISPR interference (CRISPRi) was employed for gene silencing in L. lactis NZ9000.
• Functional screening was performed to identify genes conferring bile salt resistance.

Main Results:

• The developed CRISPR-dCas9 system achieved significant gene repression, reducing gene expression by up to 99%.
• The system demonstrated effective silencing of single and multiple target genes.
• LLNZ_07335 was identified as a bile salt hydrolase, conferring bile salt resistance to L. lactis NZ9000.
• This marks the first report of a functional gene for bile salt tolerance in L. lactis.

Conclusions:

• The inducible CRISPR-dCas9 system provides a powerful new tool for gene repression in L. lactis and other LAB.
• This technology expands the genetic engineering toolkit for L. lactis, enabling advanced applications.
• The identification of LLNZ_07335 advances our understanding of bile salt tolerance mechanisms in L. lactis.