Use of CRISPR interference for efficient and rapid gene inactivation inFusobacterium nucleatum

Abstract

ABSTRACTGene inactivation via creating in-frame deletion mutations inFusobacterium nucleatumis 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-inactiveStreptococcus pyogenesCas9 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,andradD, pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline,ftsXsuppression caused filamentous cell formation akin to chromosomalftsXdeletion, while targetingradDsignificantly reduced RadD protein levels, abolishing coaggregation. The system was then extended to probe essential genesbamAandftsZ, vital for outer membrane biogenesis and cell division. Impressively,bamAsuppression disrupted membrane integrity and bacterial separation, stalling growth, whileftsZ-targeting yielded elongated cells in broth with compromised agar growth. Further studies onF. nucleatumclinical strain CTI-2 andFusobacterium periodonticumrevealed reduced indole synthesis when targetingtnaA. Moreover, silencingclpBinF. periodonticumdecreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains.IMPORTANCEHow can we effectively investigate the gene functions inFusobacterium nucleatum, 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 CRISPRi system in which dCas9 expression is controlled at the translation level by a theophylline-responsive riboswitch unit, and sgRNA expression is driven by the robust, constitutiverpsJpromoter. This approach simplifies gene inactivation in the model organism (ATCC 23726) and extends its application to previously considered resistant strains like CTI-2 andFusobacterium periodontium. With CRISPRi’s potential, it is a pivotal tool for in-depth genetic studies into fusobacterial pathogenesis, potentially unlocking targeted therapeutic strategies.