This week we profile a recent publication in Nature Communications from the laboratory of Dr. Jesse Zalatan (pictured) at UW.
Can you provide a brief overview of your lab’s current research focus?
My lab studies the physical organizing principles of biological networks in cell signaling and gene regulation, using methods ranging from mechanistic enzymology to synthetic biology. In cell signaling, we focus on the biochemical mechanisms that allow interconnected protein signaling networks to maintain specificity. In gene regulation, we are developing CRISPR-Cas tools to regulate gene expression in eukaryotic and bacterial systems, both as a discovery tool and for bioengineering applications.
What is the significance of the findings in this publication?
Our long-term goal is to use bacteria 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. To improve the output of biosynthetic pathways, we are developing new CRISPR-Cas tools to systematically and dynamically regulate multi-gene expression programs. These tools allow us to programmably target and regulate specific genomic sites. CRISPR-Cas tools to activate gene expression (CRISPRa) have been widely used in eukaryotic cells, but applications in bacterial systems remain limited.
We previously described a new bacterial CRISPRa system that enables robust, programmable activation and repression of multiple genes simultaneously with a large dynamic range of gene expression. In this paper, we describe several features of bacterial genes that impose stringent requirements on CRISPRa, and strategies for overcoming these requirements. Our most surprising finding was that there is a very narrow range of target sites that effectively activated bacterial gene expression. This and other behaviors suggest an explanation for why gene activation methods in bacteria have lagged far behind comparable tools in eukaryotic systems, where such strict target site requirements are absent.
By systematically defining the rules for effective bacterial CRISPRa sites, we have identified a clear path for improving and generalizing synthetic gene regulation in bacteria. These strategies lay the groundwork for more widespread use of bacterial CRISPRa in basic research and practical applications including functional genomics screens, metabolic engineering, and synthetic microbial communities.
This work was supervised jointly by Jesse Zalatan and James Carothers, and led by graduate students Jason Fontana and Chen Dong.
What are the next steps for this research?
This work has defined new strategies to effectively regulate gene expression in bacteria and identified fundamental new questions about gene regulation that need to be addressed to further improve the technology. Our next steps address both of these areas:
- We are applying the system we have already developed to regulate biosynthesis pathways in E. coli and P. putida bacterial strains, and are actively working to establish the system in other bacteria with useful metabolic capabilities. Our immediate biosynthesis targets are small aromatic molecules that serve as precursors to industrially-relevant thermoplastic polymers.
- We are initiating genome-wide screens and engineering new activator systems to further define the rules for effective activators and to identify new activators with broader applicability.
This work was funded by:
This work was supported by the National Science Foundation (#1817623 to J.M.C, J.G.Z. and #1844152 to J.M.C.).