(27bh) Single and Multiplexed Gene Repression in Solventogenic Clostridium Via Cas12a-Based CRISPR Interference

The Clostridium genus houses a diverse group of species which are industrially relevant based on their broad feedstock consumption capabilities and the ability to produce commodity chemicals. However, their use has been limited by their genetic intractability, as native product yields and titers are low. While strain engineering is an attractive path to improved bioproduction, the development of tools for gene perturbation and genome engineering in Clostridium has not progressed as quickly as that of other workhorse organisms. Most recently, use of CRISPR/Cas systems have been reported in Clostridium species, but their use has mostly been limited to the SpyCas9 effector as a counterselection marker or its catalytically dead mutant to reduce gene expression of target genes.

Here we have developed a dFnCas12a-based CRISPR interference system for transcriptional gene repression in mesophilic Clostridium and demonstrate its use in Clostridium acetobutylicum and Clostridium pasteurianum, achieving up to 99% reduction in transcript levels of targeted genes. We also show multiplexed repression via use of a single synthetic CRISPR array for the first time in Clostridium, achieving 99% reduction in mRNA levels of targeted genes and elucidating a unique metabolic profile for their reduced expression.

Overall, this dCas12a-based CRISPRi system provides wide targetability of Clostridium genomes and can be used to gain a better understanding of Clostridium metabolism and physiology, contributing to the development of chassis Clostridium strains for industrial use.