Use of CRISPR interference for efficient and rapid gene inactivation in .

Gene inactivation by creating in-frame deletion mutations in is time consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPR interference (CRISPRi) system. This system employs the nuclease-inactive Cas9 protein (dCas9), specifically guided to the gene of interest by a constantly expressed single-guide RNA (sgRNA). Mechanistically, this dCas9-sgRNA complex serves as an insurmountable roadblock for RNA polymerase, thus repressing the target gene transcription. Leveraging this system, we first examined two non-essential genes, and , which are pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline, suppression caused filamentous cell formation akin to chromosomal deletion, while targeting significantly reduced RadD protein levels, abolishing RadD-mediated coaggregation. The system was then extended to probe essential genes and , which are vital for outer membrane biogenesis and cell division. Impressively, suppression disrupted membrane integrity and bacterial separation, stalling growth, while targeting yielded elongated cells in broth with compromised agar growth. Further studies on clinical strain CTI-2 and revealed reduced indole synthesis when targeting . Moreover, silencing in decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains.IMPORTANCEHow can we effectively investigate the gene functions in , given the dual challenges of gene inactivation and the inherent genetic resistance of many strains? Traditional methods have been cumbersome and often inadequate. Addressing this, our work introduces a novel inducible CRISPR interference (CRISPRi) system in which dCas9 expression is controlled at the translation level by a theophylline-responsive riboswitch unit, and single-guide RNA expression is driven by the robust, constitutive promoter. This approach simplifies gene inactivation in the model organism (ATCC 23726) and extends its application to previously considered genetically intractable strains like CTI-2 and . With CRISPRi's potential, it is a pivotal tool for in-depth genetic studies into fusobacterial pathogenesis, potentially unlocking targeted therapeutic strategies.