(188d) Tunable Crispri-Based Transcriptional Control in Clostridium Pasteurianum Using dCas12a

The Clostridium genus encompasses several industrially relevant strains which are naturally capable of cellulosic and hemicellulosic biomass degradation, carbon fixation, advanced biofuel and commodity chemical production, and acting as anti-cancer therapeutics. Although advances in metabolic engineering have led to improved product tolerance, titer, yields, and feed stock consumption in several organisms, lack of metabolic engineering tools and genetic parts make metabolic engineering within the Clostridium genus challenging. To achieve optimal production yields in Clostridium, new tools must be developed for tight, predictable control of protein expression, ensuring maximum carbon flux through desired pathways by eliminating competing reactions. CRISPR interference (CRISPRi), which employs the use of a nuclease deactivated CRISPR effector, allows sequence-specific gene repression and has been used for gene perturbation in several organisms including non-model species.

Here we have developed a system for CRISPRi based repression in Clostridium using catalytically dead Cas12a (dCas12a) proteins from Moraxella bovoculi and Francisella novicida. We use a bioinformatics approach to show that these effectors are ideal for use in Clostridium based on the genus’ AT-rich genomes. We demonstrate repression in Clostridium pasteurianum, a solventogenic species able to produce a variety of chemicals including butanol from a wide range of feedstocks. We also show tunability of this system based on inducibility of dCas12a, proximity of target region to regulation elements, and strand targeted by quantifying transcription levels and solvent production. This system represents a very valuable addition to the metabolic engineering toolkit for Clostridium and would allow further progress in the optimization of various species of this genus for industrial applications.