Design Principles for Complex and Dynamically-Regulated CRISPR-Cas Gene Expression Programs in Bacteria

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This project seeks to develop new tools to regulate bacterial gene expression to support biosynthetic processes. Dramatic successes in metabolic engineering have been achieved through laborious efforts to optimize the expression of multiple genes to produce high-value chemicals. This project will develop programmable CRISPR-Cas systems to simultaneously target multiple genes for activation and repression. This will rapidly implement complex genetic programs without extensive genome engineering. Key project goals include the development of reliable control of synthetic transcriptional activators in bacteria. The implementation of these control strategies will help to dynamically regulate gene expression. 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.The goal of this proposal is to develop new tools to systematically and dynamically regulate multi-gene expression programs, and to identify regulatory architectures that can improve the output of biosynthetic pathways. CRISPR-Cas transcriptional regulatory circuits will be used to simultaneously up- and down-regulate both endogenous and heterologous genes using a combination of CRISPRi-based gene repression and CRISPRa-based synthetic activation. There are currently very few synthetic transcriptional activators available for bacterial systems, and a key component of this proposal will be to engineer new activation domains. The CRISPR-Cas system will be controlled by inducible and/or dynamically-responsive promoters to regulate the timing of multi-gene expression programs. To identify transcriptional programs that improve biosynthetic yields, RNA-based biosensors for metabolic products will be used to enable high-throughput screening of libraries of engineered strains. Combining CRISPR-Cas tools for sophisticated control of multi-gene expression programs with the ability to screen many variants will enable a systematic exploration of a large space of dynamically-responsive 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.