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Abstract TheVibrio fischeri—Euprymna scolopessymbiosis has become a powerful animal—microbe model system to examine the genetic underpinnings of symbiont development and regulation. Although there has been a number of elegant bacterial genetic technologies developed to examine this symbiosis, there is still a need to develop more sophisticated methodologies to better understand complex regulatory pathways that lie within the association. Therefore, we have developed a suite of CRISPR interference (CRISPRi) vectors for inducible repression of specific V. fischeri genes associated with symbiotic competence. The suite utilizes both Tn7-integrating and shuttle vector plasmids that allow for inducible expression of CRISPRi dCas9 protein along with single-guide RNAs (sgRNA) modules. We validated this CRISPRi tool suite by targeting both exogenous (an introduced mRFP reporter) and endogenous genes (luxCin the bioluminescence producingluxoperon, and flrA, the major regulatory gene controlling flagella production). The suite includes shuttle vectors expressing both single and multiple sgRNAs complementary to the non-template strand of multiple targeted genetic loci, which were effective in inducible gene repression, with significant reductions in targeted gene expression levels.V. fischericells harboring a version of this system targeting theluxCgene and suppressing the production of luminescence were used to experimentally validate the hypothesis that continuous luminescence must be produced by the symbiont in order to maintain the symbiosis at time points longer than the known 24-h limit. This robust new CRISPRi genetic toolset has broad utility and will enhance the study of V. fischerigenes, bypassing the need for gene disruptions by standard techniques of allelic knockout-complementation-exchange and the ability to visualize symbiotic regulation in vivo.