Abstract
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.