title: Editing of the urease gene by CRISPR-Cas in the diatomThalassiosira 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 ofThalassiosira 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 ofT. 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.
authors.affiliation: School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ UK.
authors.forename: Amanda
authors.initials: A
authors.lastname: Hopes
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH UK.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Nekrasov
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH UK.
authors.forename: Sophien
authors.initials: S
authors.lastname: Kamoun
authors.affiliation: School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ UK.
authors.forename: Thomas
authors.identifier: 0000-0001-9604-0362
authors.initials: T
authors.lastname: Mock
keywords: CRISPR-Cas; Diatom; Genome editing; Golden Gate; Thalassiosira pseudonana; Urease