Design-driven engineering of CRISPRa components and programs for rapidly optimizing multi-gene metabolic pathways

Jason Fontana 
Zalatan Lab & Carothers Lab, University of Washington

2.00 p.m. - Aula B105
Via Sommarive, 9 - Povo, Trento

Optimizing metabolic pathways to achieve high production yields often requires fine-tuning the expression level of multiple enzymes. Such efforts typically involve exploring a large combinatorial design space. To address this challenge, we are developing CRISPR-Cas activation (CRISPRa) tools for E. coli to rapidly implement combinatorial differences in the expression of multiple heterologous and endogenous genes. In this talk, I will describe our method to generate orthogonal CRISPRa-responsive synthetic promoters that can be selectively activated up to 50-fold by expressing their cognate gRNAs. We computationally designed these promoters to contain unique gRNA target sites and employed structure-based screening to identify gRNAs that avoid misfolding. We used our promoters to independently regulate the expression of multiple biosynthetic enzymes for the production of the industrial aromatic compound p-amino phenylalanine. To optimize the expression level of multiple pathway enzymes at once, we also need ways to systematically change the expression level at each promoter. We demonstrate that gene expression from our promoters can be tuned by truncating their cognate gRNAs from the 5’ end, which will allow the construction of combinatorial libraries of multi-gene expression programs. Last, I will discuss the surprisingly stringent spatial requirements for CRISPRa and how these constraints affect our ongoing efforts to activate endogenous gene expression. We hope our tools will enable us to rapidly discover new, complex multi-gene CRISPRa programs that improve production yields in metabolic programs. Jason Fontana, Chen Dong, David Sparkman-Yager, Venkata C. Chavali, Cholpisit Kiatissewee, Joanne Wong, Jesse G. Zalatan, James M. Carothers