Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

Hopes, Amanda, Nekrasov, Vladimir, Kamoun, Sophien and Mock, Thomas (2016) Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana. Plant Methods, 12 (1). ISSN 1746-4811

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    Abstract

    Background: CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas. Results: A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate. Conclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.

    Item Type: Article
    Additional Information: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
    Uncontrolled Keywords: crispr-cas,diatom,genome editing,urease,golden gate,thalassiosira pseudonana
    Faculty \ School: Faculty of Science > School of Environmental Sciences
    Faculty of Science
    ?? TSL ??
    Faculty of Science > School of Biological Sciences
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    Depositing User: Pure Connector
    Date Deposited: 25 Nov 2016 16:17
    Last Modified: 09 Apr 2019 11:43
    URI: https://ueaeprints.uea.ac.uk/id/eprint/61490
    DOI: 10.1186/s13007-016-0148-0

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