CRISPR-Cas12a-Mediated Gene Deletion and Regulation in Clostridium ljungdahlii and Its Application in Carbon Flux...

Uncovering the full potential of gas-fermenting Clostridia, attractive autotrophic bacteria capable of using synthesis gases (CO-CO₂-H₂) to produce a range of chemicals and fuels, for industrial applications relies on having efficient molecular tools for genetic modifications. Although the CRISPR-Cas9-mediated genome editing system has been developed in Clostridia, its use is limited owing to low GC content (approx. 30%) in these anaerobes. Therefore, the effector protein Cas12a, which recognizes T-rich instead of G-rich protospacer-adjacent motifs (PAMs), has evident advantages over Cas9 in CRISPR genome editing in Clostridia. Here, we developed the CRISPR-Cas12a system for efficient gene deletion and regulation in the gas-fermenting Clostridium ljungdahlii species. On the basis of screening for the most suitable Cas12a and significantly improved electrotransformation efficiency that bypassed poor repair efficiency of the Cas12a-caused DNA double-strand break (DSB) in C. ljungdahlii, efficient deletion (80-100%) of four genes (pyrE, pta, adhE1, and ctf) was achieved by using the CRISPR-FnCas12a system. Furthermore, a DNase-deactivated FnCas12a (ddCas12a) was adopted to construct a CRISPRi system to downregulate targeted genes, reaching over 80% repression for most of the chosen binding sites. This CRISPRi system was also used in a butyric acid-producing C. ljungdahlii strain to redirect carbon flux, leading to 20-40% reductions in ethanol titer that were accompanied by increased butyric acid titer. These results demonstrate the high efficiency of the CRISPR-FnCas12a system for genome engineering in C. ljungdahlii, which effectively expands the existing CRISPR-Cas toolbox in gas-fermenting Clostridium species and may play important roles in genetic manipulations where CRISPR-Cas9 is incompetent.