CRISPR/dCas-mediated counter-silencing: reprogramming dCas proteins into antagonists of xenogeneic silencers

Lsr2-like nucleoid-associated proteins act as xenogeneic silencers (XSs), repressing the expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. The targets of Lsr2-like proteins are very diverse, including prophage elements, virulence gene clusters, and biosynthetic gene clusters. Consequently, the targeted activation of XS target genes is of interest for fundamental research and biotechnological applications. Traditional methods for counter-silencing typically require promoter modifications. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach, utilizing nuclease-deficient dCas enzymes to counteract repression by Lsr2-like proteins in <i>Corynebacterium glutamicum</i> and <i>Streptomyces venezuelae</i>. The strongest effect was observed when targeting the Lsr2 nucleation site. Genome-wide transcriptome profiling revealed high specificity with minimal off-target effects. Overall, CRISPRcosi emerges as a powerful tool for the precise induction of genes silenced by xenogeneic silencers, offering new opportunities for exploring gene networks and advancing biotechnological applications.