High‐efficiency genome editing of an extreme thermophile Thermus thermophilus using endogenous type I and type III CRISPR‐Cas systems. Issue 4 (7th December 2022)

• Record Type: Journal Article
• Authors: Wang, Jinting; Wei, Junwei; Li, Haijuan; Li, Yingjun
• Is Part Of: MLife. Volume 1:Issue 4(2022)
• Page Start: 412
• Page End: 427
• Publication Date: 2022-12-07
• Subjects: endogenous CRISPR‐Cas system -- genome editing -- reporter gene -- SOD production -- Thermus thermophilus; Microbiology -- Periodicals; Microbiology; Periodicals; 579; 579
• DOI: 10.1002/mlf2.12045
• Languages: English
• ISSNs: 2770-100X

Abstract

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. Impact statement: This is the first systematic study of target interference and genome editing capabilities of endogenous clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated (Cas) protein (CRISPR‐Cas) systems of Thermus thermophilus . This study developed a high‐efficiency genome editing tool and a reporter gene system and constructed a high superoxide dismutases‐production engineered strain for T. thermophilus . Our findings provide a reference for the research and industrial application of extreme thermophiles.