2025/11/01 by Lisa Friess, Douwe van Sinderen, Ciaran M. Lee · 1 voice
Biochemistry, Genetics and Molecular Biology · #CRISPR and Genetic Engineering #Bacterial Genetics and Biotechnology #Microbial Metabolic Engineering and Bioproduction
paper · doi:10.1111/1751-7915.70260
openalex publication_date 2025/11/01 · openalex created_date 2025/11/07 · openalex updated_date 2026/07/28
This work describes the development of a CRISPR interference (CRISPRi) system for targeted gene repression in bifidobacteria. We first validated the CRISPRi-based approach using Bifidobacterium breve strains engineered to express nuclease-dead orthologs of Cas9 and demonstrated that the CRISPR-Cas system from Streptococcus thermophilus is efficient at targeting both reporter and endogenous genes through the use of single guide RNAs corresponding to the gene of interest. We also developed a one-plasmid system for targeted gene repression in bifidobacteria and demonstrated its utility by targeting genes involved in nucleotide metabolism and carbohydrate metabolism in several species of bifidobacteria. Efficient gene repression was achieved across all tested bifidobacterial species without the requirement for extensive optimization of transformation parameters or sequence optimization to avoid restriction modification systems thus removing the key barriers to genetic manipulation in this genus. This CRISPRi system provides a novel approach to functional genomics in bifidobacteria which facilitates future mechanistic studies in these commercially important microbes.