Developing a CRISPRi system with multiple inducible gRNA for orthogonal and tuneable regulation of gene expression in the fast-growing bacterium Vibrio natriegens

In the last decades, knockouts have been the way to study gene functions in many organisms. However, this approach is very time-consuming and restricted in its adjustability, as genes can only be made inoperative. Also, there is no way to gradually regulate the expression rate of target genes. In contrast, the long-term goal of this project is the implementation of a CRISPRi tool in the world’s fastest growing bacterium V. natriegens, which allows the simultaneous but independent regulation of the transcription rate of multiple genes with the help of inducible promotor-controlled gRNAs. For this, four inducible small-molecule sensors, originally optimized in E. coli (Meyer et al., 2019), were introduced in V. natriegens. Their dose-response curve, dynamic range and sensitivity were quantitatively characterized by using a fluorescent reporter gene. This way, we added a number of inducible promoters to the list of available genetic tools for V. natriegens. To utilize the newly characterized inducible promoters in our gene regulation tool, we cloned them upstream of gRNA sequences. Depending on the inducer concentrations, the gRNA’s transcription therefore was directly regulated by the small-molecular sensors allowing gradual regulation of gene expression rates over time rather than restricted switch on switch off situations of traditional knockouts. To confirm the kinetic properties of our promotor-controlled gRNA tool components, we targeted a fluorescent reporter gene with the CRISPRi system in which dCas9 and gRNA were expressed from different inducible promoters. As a result, we obtained AND-gate like characteristics, in which we only observed strong repression if both CRISPRi components were highly expressed. After having completed this initial quantitative characterization, we will apply our tool for the rapid prototyping of V. natriegens strains. We will use CRISPRi to screen for genes which can be deleted in order to increase the plasmid transformation efficiency of V. natriegens.

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