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. 2024 Aug 13;13(8):e0004024.
doi: 10.1128/mra.00040-24. Epub 2024 Jul 5.

Mobile-CRISPRi protocol optimization for Vibrionaceae

Logan J Geyman  1 , Madeline P Tanner  1 , Natalia Rosario-Meléndez  2   3 , Jason M Peters  3   4 , Mark J Mandel  2   3 , Julia C van Kessel  1
Affiliations

Mobile-CRISPRi protocol optimization for Vibrionaceae

Logan J Geyman et al. Microbiol Resour Announc. .

Abstract

Mobile clustered regularly interspaced palindromic repeats interference (Mobile-CRISPRi) is an established method for bacterial gene expression knockdown. The deactivated Cas9 protein and guide RNA are isopropyl β-D-1-thiogalactopyranoside inducible, and all components are integrated into the chromosome via Tn7 transposition. Here, we optimized methods specific for applying Mobile-CRISPRi in multiple Vibrio species.

Keywords: CRISPR; CRISPRi; Vibrio; Vibrio campbellii; Vibrio cholerae; Vibrio fischeri; Vibrio parahaemolyticus; Vibrio vulnificus; regulation of gene expression.

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Conflict of interest statement

The authors declare a conflict of interest.

Figures

Fig 1
Fig 1
(A) Schematic for Mobile-CRISPRi. P1 and P2 are both PLlacO1 (6). (B) Conjugative efficiency of plasmid pLG2 targeting luxR in V. campbellii with and without RP4 helper plasmid pRK600 (3). The line in each box represents the group mean. Each point is a biological replicate that is the average of two technical replicates. Statistical analysis performed using GraphPad Prism: unpaired t-test on log-transformed data. ***P = 0.0005. (C) Table outlining the conjugation resources available for this method. *pEVS104 was not the helper used in panel B.
See this image and copyright information in PMC

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