Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae

Vibrio cholerae O1 El Tor , the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools exist. While CRISPRi has been applied in V. cholerae , improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium. In this study, we introduce a pooled genome wide CRISPRi library construction specifically optimized for this V. cholerae strain, characterized by minimal cytotoxicity and streamlined experimental setup. This library allows the depletion of 3, 674 (98.9%) annotated genes from the V. cholerae genome. To confirm its effectiveness, we screened for essential genes during exponential growth in rich medium and identified 368 genes for which guides were significantly depleted from the library (log2FC < - 2). Remarkably, 82% of these genes had previously been described as hypothetical essential genes in V. cholerae or in a closely related bacterium, V. natriegens . We thus validated the robustness and accuracy of our CRISPRi-based approach for assessing gene fitness in a given condition. Our findings highlight the efficacy of the developed CRISPRi platform as a powerful tool for high-throughput functional genomics studies of V. cholerae .