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

Amanda Hopes, Vladimir Nekrasov, View ORCID ProfileSophien Kamoun, View ORCID ProfileThomas Mock
doi: https://doi.org/10.1101/062026
Amanda Hopes
1School of Environmental Science, Universityof East Anglia, Norwich Research Park, NR4 7TJ, Norwich, UK.
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Vladimir Nekrasov
2The Sainsbury Laboratory, Norwich Research Park, NR4 7UH, Norwich, UK
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Sophien Kamoun
2The Sainsbury Laboratory, Norwich Research Park, NR4 7UH, Norwich, UK
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  • ORCID record for Sophien Kamoun
Thomas Mock
1School of Environmental Science, Universityof East Anglia, Norwich Research Park, NR4 7TJ, Norwich, UK.
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  • ORCID record for Thomas Mock
  • For correspondence: t.mock@uea.ac.uk
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Abstract

Background: CRISPR-Cas is a recent and powerful edition 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 T. pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.

Results: A single construct wa assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37nt 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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted July 18, 2016.
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Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana
Amanda Hopes, Vladimir Nekrasov, Sophien Kamoun, Thomas Mock
bioRxiv 062026; doi: https://doi.org/10.1101/062026
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Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana
Amanda Hopes, Vladimir Nekrasov, Sophien Kamoun, Thomas Mock
bioRxiv 062026; doi: https://doi.org/10.1101/062026

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