Mobile-CRISPRi as a powerful tool for modulatingVibriogene expression

Abstract

AbstractCRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducibledcas9andsgRNAgenes into the genome using Tn7transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multipleVibriospecies:Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, andVibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used inVibriospecies to systematically probe gene function and essentiality in various behaviors and native environments.