Challenges and Opportunities with CRISPR Activation in Bacterial and Cell-Free Pathway Engineering (Invited Speaker)

The development of CRISPR-Cas tools for rapidly programming gene expression promises to accelerate metabolic pathway engineering. The application of CRISPR-Cas tools for transcriptional repression (CRISPRi) in bacterial metabolic engineering is well-established. By comparison, the creation of CRISPR-Cas tools for transcriptional activation (CRISPRa) in bacteria has lagged. Together with our collaborators, we have developed new tools for bacterial CRISPRa by 1) identifying effective transcriptional activators, 2) systematically defining rules for effective bacterial CRISPRa, and 3) creating approaches for encoding complex functions in engineered guide RNAs. We have shown that these CRISPRa tools can drive expression of multi-gene biosynthetic pathways from multiple promoters and in multiple hosts, including E. coli, P. putida and E. coli-derived cell-free TXTL. In this presentation, I will describe our most recent results and outline strategies for integrating CRISPRa and CRISPRi into data-driven workflows to create new capabilities for rapidly optimizing pathways in bacterial and cell-free bioproduction systems.