Gene Silencing by CRISPR Interference in Fusobacterium nucleatum.

Fusobacterium nucleatum is a strictly anaerobic bacterium associated with periodontal disease and several systemic conditions, including colorectal cancer and adverse pregnancy outcomes. Genetic manipulation in F. nucleatum has been limited by poor transformation efficiency and difficulty studying essential genes. To overcome these challenges, we developed a CRISPR interference (CRISPRi) system that enables efficient and reversible gene silencing without altering the genome. This system uses an inducible dCas9 and a customizable sgRNA expressed from a pCWU6-based shuttle plasmid (pZP4C). In this chapter, we present a step-by-step protocol for designing sgRNAs, constructing CRISPRi plasmids, transforming F. nucleatum ATCC 23726, and evaluating gene silencing phenotypes. We use the nonessential gene ftsW, which encodes a protein required for peptidoglycan synthesis and cell division, as a model target. This protocol is also applicable to other genetically recalcitrant F. nucleatum strains, offering a versatile tool for investigating both essential and nonessential gene functions.