@article {Hopes062026, author = {Amanda Hopes and Vladimir Nekrasov and Sophien Kamoun and Thomas Mock}, title = {Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana}, elocation-id = {062026}, year = {2016}, doi = {10.1101/062026}, publisher = {Cold Spring Harbor Laboratory}, 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.}, URL = {https://www.biorxiv.org/content/early/2016/07/18/062026}, eprint = {https://www.biorxiv.org/content/early/2016/07/18/062026.full.pdf}, journal = {bioRxiv} }