ArticleACS synthetic biology2017
Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9.
Article in ACS synthetic biology, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 34 papers.
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Who cites it
34 citing papers in PubMed.
- Elevated temperature undermines the efficacy ofmSphere · 2026Article
- An inducer-independent, single-plasmid CRISPR-Cas9 system for genome editing in Bacillus species.Applied microbiology and biotechnology · 2026Article
- Development of a markerless tool for targeted chromosome modification in the thermophilic and methylotrophic bacterium Bacillus methanolicus.Microbial cell factories · 2025Article
- A thermostable Cas9-based genome editing system for thermophilic acetogenic bacteriumApplied and environmental microbiology · 2025Article
- The engineering toolbox of Parageobacillus thermoglucosidasius.Applied microbiology and biotechnology · 2025Review
- CRISPR-Cas9-driven genome editing inFrontiers in microbiology · 2025Article
- A highly efficient method for genomic deletion across diverse lengths in thermophilicSynthetic and systems biotechnology · 2024Article
- Characterization of the AcrIIC1 anti‒CRISPR protein for Cas9‒based genome engineering in E. coli.Communications biology · 2023Article
- CRISPR-Based Gene Editing inPharmaceuticals (Basel, Switzerland) · 2023Review
- Single-Stranded DNA-Binding Proteins Mediate DSB Repair and Effectively Improve CRISPR/Cas9 Genome Editing inMicroorganisms · 2023Article
- Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production ofiScience · 2023Article
- Genome-scale reconstruction and metabolic modelling of the fast-growing thermophileMetabolic engineering communications · 2022Article
- Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages.Journal of applied microbiology · 2022Review
- Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool inMicroorganisms · 2022Article
- Bacterial genome reductions: Tools, applications, and challenges.Frontiers in genome editing · 2022Review
- Gene Editing Technologies for Sugarcane Improvement: Opportunities and Limitations.Sugar tech : an international journal of sugar crops & related industries · 2022Article
- Temperature-Inducible Precision-Guided Sterile Insect Technique.The CRISPR journal · 2021Article
- Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant.Scientific reports · 2021Article
- Role of extremophiles and their extremozymes in biorefinery process of lignocellulose degradation.Extremophiles : life under extreme conditions · 2021Review
- Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles.The CRISPR journal · 2021Article
Corrections and comments
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Authors and funding
7 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45-55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.
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