Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles

The ability of CRISPR-Cas12a nucleases to function reliably in a wide range of species has been key to their rapid adoption as genome engineering tools. However, so far, Cas12a nucleases have been limited for use in organisms with growth temperatures up to 37 °C. Here, we biochemically characterize three Cas12a orthologs for their temperature stability and activity. We demonstrate that Francisella novicida Cas12a (FnCas12a) has great biochemical potential for applications that require enhanced stability, including use at temperatures >37°C. Furthermore, by employing the moderate thermophilic bacterium Bacillus smithii as our experimental platform, we demonstrate that FnCas12a is active in vivo at temperatures up to 43°C. Subsequently, we develop a single-plasmid FnCas12a-based genome editing tool for B. smithii, combining the FnCas12a targeting system with plasmid-borne homologous recombination (HR) templates that carry the desired modifications. Culturing of B. smithii cells at 45°C allows for the uninhibited realization of the HR-based editing step, while a subsequent culturing step at reduced temperatures induces the efficient counterselection of the non-edited cells by FnCas12a. The developed gene-editing tool yields gene-knockout mutants within 3 days, and does not require tightly controllable expression of FnCas12a to achieve high editing efficiencies, indicating its potential for other (thermophilic) bacteria and archaea, including those with minimal genetic toolboxes. Altogether, our findings provide new biochemical insights into three widely used Cas12a nucleases, and establish the first Cas12a-based bacterial genome editing tools for moderate thermophilic microorganisms.