PT  - JOURNAL ARTICLE
AU  - Amanda Hopes
AU  - Vladimir Nekrasov
AU  - Sophien Kamoun
AU  - Thomas Mock
TI  - Editing of the urease gene by CRISPR-Cas in the diatom <em>Thalassiosira pseudonana</em>
AID  - 10.1101/062026
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 062026
4099  - http://biorxiv.org/content/early/2016/07/18/062026.short
4100  - http://biorxiv.org/content/early/2016/07/18/062026.full
AB  - 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.