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

Abstract

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.

A highly efficient method for genomic deletion across diverse lengths in thermophilic Parageobacillus thermoglucosidasius

“…1 d and e). Considering the widespread utilization of endogenous or heterogenous type I and II systems in thermophiles [ 16 , 20 , [34] , [35] , [36] ], our discoveries provide essential components for developing extensive range genomic deletion tools or donor DNA-free gene knockout tools in other biotechnological potential thermophilic strains [ 37 ].…”

Application of functional genomics for domestication of novel non-model microbes

“…As it currently stands, there is a lack of broadly applicable genetic tools, including selectable markers, promoters, plasmid origins, and transformation protocols, as broadly utilized mesophilic tools are not functional in these thermophilic microbes (Y. Wang et al., 2024 ; Ye et al., 2023 ). While there are a growing number of tools being generated for use in thermophilic microbes (Adalsteinsson et al., 2021 ; Le & Sun, 2022 ; Riley et al., 2019 ; Walker et al., 2020 ; Wang et al., 2022 ; Wu et al., 2023 ; Yang et al., 2023 ), developing new tools and implementing CRISPR mediated genome engineering and/or thermostable serine recombinase-assisted genome engineering (SAGE) mediated integration would greatly simplify testing production of various industrial chemicals in these currently underutilized microbes (Fenster & Eckert, 2021 ; Wu et al., 2023 ). Continued decrease in -omics costs coupled with advanced genome modification techniques makes adopting extremophiles and other novel, less studied microbes as industrial chassis a viable and exciting avenue of research that should be more intensely pursued.…”

Nano-Biofertilizer Formulations for Agriculture: A Systematic Review on Recent Advances and Prospective Applications

“…They relieve the effects of environmental stressors by controlling the distribution of nutrients, moving them to their intended destination. Their most notable qualities are a lower need for synthetic fertilizers, higher yields with less time and effort, lower environmental impacts, lower costs, and more nutrient availability and absorption [ 18 ]. It was shown that NPs can affect plant–microbe interactions in two ways: directly, by altering the accessibility to nutrients in the rhizospheric soil, and indirectly, by stimulating microorganisms.…”