Post-transcriptional genome regulation in bacteria with next generation CRISPR-Cas tools

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This project seeks to develop new tools to regulate bacterial gene expression for biosynthesis. Dramatic successes in metabolic engineering have been achieved through laborious efforts to optimize gene expression for the production of high-value chemicals. Programmable, DNA-targeting CRISPR-Cas tools can be used to rapidly implement complex genetic programs, but in bacteria these systems have limitations in their ability to precisely control individual genes. To improve the ability to precisely control cellular behavior, the investigators will develop RNA-targeting CRISPR-Cas systems that act post-transcriptionally and may overcome the limitations of the DNA-targeting systems. The sophisticated control systems developed in this project will be useful for practical biosynthesis, bacterial engineering, and basic research in bacteria. These findings will be incorporated into educational materials and courses taught to chemistry and engineering students. This project will also provide opportunities for underrepresented students at the high school and undergraduate levels to participate in laboratory research.Bacterial metabolic pathways perform complex chemical transformations with high specificity to produce biosynthetic products. Introducing heterologous genes allows metabolism to be diverted to new synthetic targets, but optimizing the function of these engineered strains is challenging. The goal of this proposal is to develop a new class of bacterial RNA-targeting tools to systematically regulate multi-gene expression programs, and to identify regulatory architectures that can improve the output of biosynthetic pathways. Previously, DNA-targeting CRISPR-Cas transcriptional regulatory circuits have been successfully assembled into sophisticated multi-gene regulatory programs. Despite their enormous potential, DNA-targeting CRISPR-Cas systems have important limitations in their ability to precisely up- or down-regulate individual bacterial gene targets. For this project, the investigators will create dCas13-based RNA-targeting tools, which act post-transcriptionally and may overcome the limitations of DNA-targeting CRISPR-Cas systems. The immediate goal of the project is to develop new capabilities for gene repression and activation at the translational level. First, the investigators will use Agile BioFoundry (ABF) capabilities to learn global rules for dCas13-mediated translational regulation of gene expression and metabolic flux in bacteria. Next, they will systematically compare dCas13-mediated translational regulation to dCas9-mediated transcriptional control to determine whether these systems can produce distinct functional effects. Finally, they will demonstrate the utility of this knowledge by implementing translational control systems in Design-Build-Test-Learn (DBTL) cycles applied to engineer aromatic biosynthesis in a non-model microbe that can be used for industrial bioproduction. These systems will further expand the toolkit for exploring the large space of regulatory architectures to optimize bacterial biosynthesis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.