Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum

Gene inactivation via creating in-frame deletion mutations in Fusobacterium nucleatum is time-consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPRi system. This system employs the nuclease-inactive Streptococcus pyogenes 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, ftsX, and radD , pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline, ftsX suppression caused filamentous cell formation akin to chromosomal ftsX deletion, while targeting radD significantly reduced RadD protein levels, abolishing coaggregation. The system was then extended to probe essential genes bamA and ftsZ , vital for outer membrane biogenesis and cell division. Impressively, bamA suppression disrupted membrane integrity and bacterial separation, stalling growth, while ftsZ- targeting yielded elongated cells in broth with compromised agar growth. Further studies on F. nucleatum clinical strain CTI-2 and Fusobacterium periodonticum revealed reduced indole synthesis when targeting tnaA . Moreover, silencing clpB in F. periodonticum decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains.