Characterizing a thermostable Cas9 for bacterial genome editing and silencing
| Autor: | Elleke Fenna Bosma, Richard van Kranenburg, Ioannis Mougiakos, Mihris Ibnu Saleem Naduthodi, Prarthana Mohanraju, John van der Oost, Valentijn Vrouwe, Max Finger Bou, Alex Gussak, Rudolf B. L. Brinkman |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Bio Process Engineering Hot Temperature Science 030106 microbiology General Physics and Astronomy Bacillus Computational biology Bacterial genome size Biology Microbiology General Biochemistry Genetics and Molecular Biology Article Genome engineering 03 medical and health sciences Research & Innovation Bacterial Proteins Microbiologie Enzyme Stability Gene silencing Life Science Gene Silencing lcsh:Science VLAG Subgenomic mRNA Gene Editing Genetics Dean & Managers Office Multidisciplinary Corporate Education Research & Innovation Pseudomonas putida Cas9 Thermophile Geobacillus General Chemistry Endonucleases biology.organism_classification Family member 030104 developmental biology lcsh:Q Corporate Education Genome Bacterial Bacteria |
| Zdroj: | Nature Communications 8 (2017) 1 Nature Communications Nature Communications, Vol 8, Iss 1, Pp 1-11 (2017) Nature Communications, 8(1) Mougiakos, I, Mohanraju, P, Bosma, E F, Vrouwe, V, Bou, M F, Naduthodi, M I S, Gussak, A, Brinkman, R B L, van Kranenburg, R & van der Oost, J 2017, ' Characterizing a thermostable Cas9 for bacterial genome editing and silencing ', Nature Communications, vol. 8, 1647 . https://doi.org/10.1038/s41467-017-01591-4 |
| ISSN: | 2041-1723 |
| Popis: | CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range. CRISPR-Cas9 genome engineering tools have found wide application in a range of species, however they are unsuitable for applications at elevated temperatures. Here the authors characterise ThermoCas9 from which is functional from 20°C to 70°C. |
| Databáze: | OpenAIRE |
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