Zink, IA;
Fouqueau, T;
Tarrason Risa, G;
Werner, F;
Baum, B;
Bläsi, U;
Schleper, C;
(2020)
Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing.
RNA Biology
10.1080/15476286.2020.1813411.
(In press).
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Comparative CRISPR type III based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing.pdf - Published Version Download (5MB) | Preview |
Abstract
CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific response to invading nucleic acids. Therefore, these systems can be harnessed for gene knockdown technologies even in hyperthermophilic archaea to study essential genes. We show here the broader usability of this posttranscriptional silencing technology by expanding the application to further essential genes and systematically analysing and comparing silencing thresholds and escape mutants. Synthetic guide RNAs expressed from miniCRISPR cassettes were used to silence genes involved in cell division (cdvA), transcription (rpo8), and RNA metabolism (smAP2) of the two crenarchaeal model organisms Saccharolobus solfataricus and Sulfolobus acidocaldarius. Results were systematically analysed together with those obtained from earlier experiments of cell wall biogenesis (slaB) and translation (aif5A). Comparison of over 100 individual transformants revealed gene-specific silencing maxima ranging between 40 and 75%, which induced specific knockdown phenotypes leading to growth retardation. Exceedance of this threshold by strong miniCRISPR constructs was not tolerated and led to specific mutation of the silencing miniCRISPR array and phenotypical reversion of cultures. In two thirds of sequenced reverted cultures, the targeting spacers were found to be precisely excised from the miniCRISPR array, indicating a still hypothetical, but highly active recombination system acting on the dynamics of CRISPR spacer arrays. Our results indicate that CRISPR type III - based silencing is a broadly applicable tool to study in vivo functions of essential genes in Sulfolobales which underlies a specific mechanism to avoid malignant silencing overdose.
| Type: | Article |
|---|---|
| Title: | Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1080/15476286.2020.1813411 |
| Publisher version: | http://dx.doi.org/10.1080/15476286.2020.1813411 |
| Language: | English |
| Additional information: | © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. |
| Keywords: | Sulfolobales, CRISPR type III, archaea, essential genes, hyperthermophilic, knockdown |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10111470 |
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