ArticleScientific reports2021
Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant.
Article in Scientific reports, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed, 58 citations in OpenAlex.
- Genome editing of phylogenetically distinct bacteria using cross-species retron-mediated recombineering.Nature biotechnology · 2026Article
- Sugar-inducible promoters for manipulation of core metabolic pathways in the thermophilic acetogenApplied and environmental microbiology · 2025Article
- A thermostable Cas9-based genome editing system for thermophilic acetogenic bacteriumApplied and environmental microbiology · 2025Article
- Construction of primary chassis cells with efficient protein expression in Thermus thermophilus.Microbial cell factories · 2025Article
- Designing thermophilic, synthetic microbial communities for consolidated bioprocessing.Biodesign research · 2025Review
- Emerging trends in genome integration tools for precision engineering of diverse bacterial species.Synthetic biology (Oxford, England) · 2025Review
- Exploitation of Biodiversity in Bioeconomy: Examples, Opportunities, and Challenges.Advances in biochemical engineering/biotechnology · 2025Review
- Biofuel production from lignocellulose via thermophile-based consolidated bioprocessing.Engineering microbiology · 2024Review
- Thermostable in vitro transcription-translation compatible with microfluidic droplets.Microbial cell factories · 2024Article
- TemStaPro: protein thermostability prediction using sequence representations from protein language models.Bioinformatics (Oxford, England) · 2024Article
- Application of functional genomics for domestication of novel non-model microbes.Journal of industrial microbiology & biotechnology · 2024Review
- Expression and Functional Analysis of the Compact ThermophilicInternational journal of molecular sciences · 2023Article
- Genomic Insights on the Carbon-Negative Workhorse: Systematical Comparative Genomic Analysis on 56Bioengineering (Basel, Switzerland) · 2023Article
- A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering.Nature communications · 2023Article
- Metabolic engineering of Thermoanaerobacterium AK17 for increased ethanol production in seaweed hydrolysate.Biotechnology for biofuels and bioproducts · 2023Article
- Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering.Microbial biotechnology · 2023Article
- Immunomodulation-a general review of the current state-of-the-art and new therapeutic strategies for targeting the immune system.Frontiers in immunology · 2023Review
- Article
- Fungal Proteases as Emerging Biocatalysts to Meet the Current Challenges and Recent Developments in Biomedical Therapies: An Updated Review.Journal of fungi (Basel, Switzerland) · 2022Review
- Deletion of the primase-polymerases encoding gene, located in a mobile element inFrontiers in microbiology · 2022Article
Corrections and comments
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Authors and funding
8 authors at 5 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Thermophilic organisms are extensively studied in industrial biotechnology, for exploration of the limits of life, and in other contexts. Their optimal growth at high temperatures presents a challenge for the development of genetic tools for their genome editing, since genetic markers and selection substrates are often thermolabile. We sought to develop a thermostable CRISPR-Cas9 based system for genome editing of thermophiles. We identified CaldoCas9 and designed an associated guide RNA and showed that the pair have targetable nuclease activity in vitro at temperatures up to 65 °C. We performed a detailed characterization of the protospacer adjacent motif specificity of CaldoCas9, which revealed a preference for 5'-NNNNGNMA. We constructed a plasmid vector for the delivery and use of the CaldoCas9 based genome editing system in the extreme thermophile Thermus thermophilus at 65 °C. Using the vector, we generated gene knock-out mutants of T. thermophilus, targeting genes on the bacterial chromosome and megaplasmid. Mutants were obtained at a frequency of about 90%. We demonstrated that the vector can be cured from mutants for a subsequent round of genome editing. CRISPR-Cas9 based genome editing has not been reported previously in the extreme thermophile T. thermophilus. These results may facilitate development of genome editing tools for other extreme thermophiles and to that end, the vector has been made available via the plasmid repository Addgene.
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