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

Amanda Hopes; Vladimir Nekrasov; Sophien Kamoun; Thomas Mock

doi:10.1186/s13007-016-0148-0

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

Amanda Hopes, Vladimir Nekrasov, Sophien Kamoun, Thomas Mock

Research output: Contribution to journal › Article › peer-review

116 Citations (Scopus)

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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.

Original language	English
Article number	49
Journal	Plant Methods
Volume	12
Issue number	1
DOIs	https://doi.org/10.1186/s13007-016-0148-0
Publication status	Published - 24 Nov 2016

Keywords

CRISPR-Cas
Diatom
Genome editing
Urease
Golden Gate
Thalassiosira pseudonana

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Sophien Kamoun
- S.Kamounuea.acuk
- School of Biological Sciences - Professor of Biology
- Plant Sciences - Member
Person: Research Group Member, Academic, Teaching & Research
Thomas Mock
- T.Mockuea.acuk
- School of Environmental Sciences - Professor of Marine Microbiology
- Centre for Ecology, Evolution and Conservation - Member
- Centre for Ocean and Atmospheric Sciences - Member
- Environmental Biology - Member
- ClimateUEA - Member
Person: Member, Research Group Member, Research Centre Member, Academic, Teaching & Research

From the North Sea to the Arctic Ocean; The Impact of Temperature on Eukaryotic Phytoplankton.
Mock, T., Moulton, V., Toseland, A., Toseland, A., Utting, R. & Utting, R.
Natural Environment Research Council
29/07/13 → 28/07/16
Project: Research

Cite this

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title = "Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana",

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.",

keywords = "CRISPR-Cas, Diatom, Genome editing, Urease, Golden Gate, Thalassiosira pseudonana",

author = "Amanda Hopes and Vladimir Nekrasov and Sophien Kamoun and Thomas Mock",

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PY - 2016/11/24

Y1 - 2016/11/24

N2 - 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.

AB - 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.

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

Abstract

Keywords

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Profiles

Sophien Kamoun

Thomas Mock

Projects

From the North Sea to the Arctic Ocean; The Impact of Temperature on Eukaryotic Phytoplankton.

Cite this