CRISPR interference strategies for studies in essential gene function and live cell fluorescent imaging of DNA elements in Escherichia coli

Abstract

<p>Traditional gene knockout methods are incompatible for studying essential gene function because inactivation would lead to cell death, so alternative strategies requiring the construction or isolation of conditional lethal mutants are used to observe phenotypic effects during gene depletion. These methods are technically demanding to engineer, which has limited characterization of essential genes in Escherichia coli. There is a need for better genetic tools to interrogate essential gene targets and one promising technique, known as CRISPR interference (CRISPRi), provides many advantages over existing methods. CRISPRi utilizes a nuclease-inactivated Cas9 (dCas9) and a programmable guide-RNA to bind to targeted DNA sequences and disrupt transcription, resulting in knockdown of gene expression levels. The work presented in this thesis describes several strategies that enable the optimization of CRISPRi for conditional repression of essential genes in E. coli. Using a modular design approach, a series of inducible dCas9 vectors were constructed that allow for rapid single-copy integration into the chromosome to achieve tightly-regulated expression. To refine the control of the CRISPRi system, a microfluidic platform was developed to generate inducer chemical-gradients for “tuning” targeted essential gene expression to desired levels of transcriptional strength. To expand the versatility of the CRISPRi, the system was repurposed as a tool to visualize localization of DNA elements by targeted binding of dCas-fluorophore fusions, a method that has not been effectively optimized in E. coli. Using these approaches, we demonstrate how CRISPRi can be applied to interrogate essential gene functions of protein export, showing that modifications to model protein substrates can alter inner membrane insertion through depletion of the Sec and YidC pathways. Next, microfluidic control of dCas9-mediated silencing showed that incremental repression of metG can induce cells into an antibiotic persistent state, leading to increased survival during ampicillin exposure. Finally, we show the effectiveness of CRISPRi as a live-fluorescent imaging method, enabling real-time observation of DNA localization and dynamics of plasmid elements and the chromosome. The collective results of this work provide a roadmap for applying CRISPRi to demanding research topics that include future studies addressing the role of tRNA synthetases in antibiotic persistence, and the construction of a live-imaging method that requires minimal genetic modification to the bacterial host.</p>