High-efficiency genome editing of an extreme thermophile Thermus thermophilus using endogenous type Ⅰ and type Ⅲ CRISPR-Cas systems

Thermus thermophilus is an attractive species in the bioindustry due to its valuable natural products, abundant thermophilic enzymes, and promising fermentation capacities. However, efficient and versatile genome editing tools are not available for this species. In this study, we developed an efficient genome editing tool for T. thermophilus HB27 based on its endogenous type Ⅰ-B, Ⅰ-C, and Ⅲ-A/B CRISPR-Cas systems. First, we systematically characterized the DNA interference capabilities of the different types of the native CRISPR-Cas systems in T. thermophilus HB27. We found that genomic manipulations such as gene deletion, mutation, and in situ tagging could be easily implemented by a series of genome-editing plasmids carrying an artificial self-targeting mini-CRISPR and a donor DNA responsible for the recombinant recovery. We also compared the genome editing efficiency of different CRISPR-Cas systems and the editing plasmids with donor DNAs of different lengths. Additionally, we developed a reporter gene system for T. thermophilus based on a heat-stable β-galactosidase gene TTP0042, and constructed an engineered strain with a high production capacity of superoxide dismutases by genome modification.