Single and multiplexed gene repression in solventogenic Clostridium via Cas12a-based CRISPR interference

The Gram-positive,spore-forming,obligate anaerobic firmicute species that make up the Clostridium genus have broad feedstock consumption capabilities and produce value-added metabolic products,but genetic manipulation is difficult,limiting their broad appeal.CRISPR-Cas systems have recently been applied to Clostridium species,primarily using Cas9 as a counterselection marker in conjunction with plasmid-based homologous recombination.CRISPR interference is a method that reduces gene expression of specific genes via precision targeting of a nuclease deficient Cas effector protein.Here,we develop a dCas12a-based CRISPR interference system for transcriptional gene repression in multiple mesophilic Clostridium species.We show the Francisella novicida Cas12a-based system has a broader applicability due to the low GC content in Clostridium species compared to CRISPR Cas systems derived from other bacteria.We demonstrate>99%reduction in transcript levels of targeted genes in Clostridium acetobutylicum and>75%reduction in Clostridium pasteurianum.We also demonstrate multiplexed repression via use of a single synthetic CRISPR array,achieving 99%reduction in targeted gene expression and elucidating a unique metabolic profile for their reduced expression.Overall,this work builds a foundation for high throughput genetic screens without genetic editing,a key limitation in current screening methods used in the Clostridium community.