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.
CITATION STYLE
Hopes, A., Nekrasov, V., Kamoun, S., & Mock, T. (2016). Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana. Plant Methods, 12(1). https://doi.org/10.1186/s13007-016-0148-0
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