Dynamic regulation of metabolic pathways with CRISPR-dCAS9 toehold-gated rna regulators

Abstract

In metabolic engineering, once a metabolic pathway is expressed, there is an optimization process that aims to increase the production, titer, rate, and yield of the target molecule with a goal to direct the maximum possible amount of metabolic flux toward the pathway of interest. Our labs were able to take the dCas9 system and incorporate the spacer into a modified toehold switch creating a toehold-gated sgRNA (thgRNA) which acts as an orthogonal, transcriptional regulator when activated by an endogenous trigger strand. This system was used to show transcriptional repression of simple and complex metabolic pathways in Escherichia Coli (E. coli). We also looked at a variety of factors including: promoter strength, temperature, and induction time on mCherry fluorescence and violacein production. Previously, we introduced the violacein pathway, a five-step metabolic pathway, into a pETM6 vector with weakened T7 promoter. By incorporating spacers targeting these weakened promoters into thgRNA, we were able to show repression of individual and multiple genes. Our thgRNA are able to function as a conditional activation of CRISPR-based systems by using highly predictable toehold-mediated strand displacement reactions. Sequence specific unblocking of the spacer allows for both orthogonality and low cross-talk between thgRNA. Additionally, these devices do not require the screening of large libraries currently needed to create such specific riboregulators as the CRISPR spacers are so sequence specific.