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A CRISPRi Gene Regulation System for Bifidobacteria.
Lisa Friess1,2, Douwe van Sinderen1,2, Ciaran Lee1,3
1APC Microbiome Ireland, University College Cork, Cork, Ireland.
Microbial Biotechnology
|November 7, 2025
Summary
Researchers developed a CRISPR interference (CRISPRi) system for gene repression in bifidobacteria. This efficient system simplifies genetic manipulation for studying these important microbes.
Area of Science:
- Microbiology
- Molecular Biology
- Genetic Engineering
Background:
- Bifidobacteria are crucial commercially important microbes.
- Genetic manipulation in bifidobacteria is challenging.
- CRISPR interference (CRISPRi) offers a potential solution for gene repression.
Purpose of the Study:
- To develop and validate a CRISPRi system for targeted gene repression in bifidobacteria.
- To establish a one-plasmid system for efficient genetic manipulation in this genus.
- To facilitate functional genomics studies in bifidobacteria.
Main Methods:
- Engineered Bifidobacterium breve strains with nuclease-dead Cas9 orthologs.
- Utilized CRISPR-Cas system from Streptococcus thermophilus with single guide RNAs.
- Developed a one-plasmid system for gene repression.
- Targeted genes involved in nucleotide and carbohydrate metabolism.
Main Results:
- Validated CRISPRi system for targeting reporter and endogenous genes in Bifidobacterium breve.
- Demonstrated efficient gene repression across multiple bifidobacterial species.
- Achieved successful repression without extensive optimization of transformation or sequence parameters.
- Overcame key barriers to genetic manipulation in bifidobacteria.
Conclusions:
- The developed CRISPRi system is efficient and versatile for gene repression in bifidobacteria.
- This system simplifies genetic manipulation, removing previous limitations.
- Provides a novel tool for functional genomics and mechanistic studies in commercially important bifidobacteria.
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