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. While this potential has begun to be realized industrially, widespread use and study of Clostridium has been stunted compared to workhorse organisms in large part due to its members being mostly obligate anaerobes and its genetic recalcitrance. Strain engineering with existing tools is time-consuming, labor-intensive and does not enable high-throughput screening. For more efficient studying and engineering of Clostridium, more tools must be developed which enable rapid genotype to phenotype mapping, as well as, perturbation of gene expression for tight and predictable control of protein production. Here we demonstrate gene repression in solventogenic Clostridium species through use of a nuclease-deactivated Francisella novicida Cas12a (dFnCas12a) based CRISPR interference system. The recognition of a T-rich protospacer adjacent motif (PAM) site makes dFnCas12a suited for use in organisms with A/T-rich genomes like Clostridium spp. We show the construction and use of a single-plasmid dFnCas12a CRISPRi system which utilizes a lactose inducible promoter for controlled expression of the dFnCas12a gene and a single-step golden gate assembly for the insertion of multiple target sequences into the crRNA locus. We achieve over 90% transcriptional-level repression of targeted genes in C. pasteurianum (Cpa) and C. acetobutylicum (Cac). We use our CRISPRi system to eliminate solvent production in Cac through targeted repression of the spo0A gene. Metabolite analysis via liquid chromatography shows this redistribution of carbon flux in undetectable levels of butanol and acetone and increased butyrate production. We describe the simultaneous repression of multiple genes through the expression of a multiplexed CRISPR array. We show rapid construction of duplexed CRISPR arrays via annealed 72-nucleotide oligos, and the potential to insert any number of targets. We apply this system in Cpa to demonstrate its applicability across Clostridium species, targeting the 1,3-propanediol pathway and hydrogen production. We will also discuss progress toward serial genetic editing using an adapted version of the multiplexed Cas12a system described here.

Altering metabolic flux in Clostridium acetobutylicum via Cas12a based CRISPRi and genome editing methods

ACS Spring 2022

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