CRISPR-Cas activation for regulating multi-gene expression in bacteria

Abstract

Bacterial metabolism is comprised of large and complex gene networks that can produce valuable chemical products. In principle, these networks can be reengineered using synthetic multi-gene transcriptional programs to optimize production of high-value compounds. However, our limited ability to regulate multi-gene expression with precision makes this goal difficult to achieve. While programmable gene repression in bacteria is well-established, there is a lack of robust tools for programmable gene activation. This work describes our efforts to develop new tools for bacteria to independently target and predictably manipulate the expression levels of multiple genes. We developed a programmable CRISPR-Cas transcriptional activation (CRISPRa) system for E. coli that uses modified guide RNAs to recruit a transcriptional activator. When used in concert with CRISPRi, our tools can be used to simultaneously activate and repress multiple genes. However, to expand our ability to regulate synthetic and endogenous pathways, we need predictive rules for selecting effective target sites for CRISPRa. To uncover these rules, we systematically investigated the requirements for CRISPRa in bacteria. In contrast to comparable systems in eukaryotes, we show that bacterial CRISPRa is sensitive to the geometry of the interaction between the CRISPRa complex and the synthetic promoter. Last, we discuss engineering dynamically regulated CRISPR-Cas systems to control the timing of the expression of multiple genes. These next-generation tools will enable us to engineer sophisticated multi-gene transcriptional networks that more closely resemble natural gene networks. Together, our work provides a foundation toward rapidly discovering and implementing new multi-gene programs that improve production of high-value chemicals.