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bioRxiv · 10.1101/2024.01.17.575898

Mobile-CRISPRi as a powerful tool for modulating Vibrio gene expression

Geyman, L. J.·Tanner, M.·Rosario-Melendez, N.·Peters, J.·Mandel, M. J.·van Kessel, J. C.
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Abstract

CRISPRi (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-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio 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 in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.

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Geyman, L. J., Tanner, M., Rosario-Melendez, N., Peters, J., Mandel, M. J., van Kessel, J. C.. 2024-01-18. Mobile-CRISPRi as a powerful tool for modulating Vibrio gene expression. https://doi.org/10.1101/2024.01.17.575898

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