Journal
PLANT METHODS
Volume 12, Issue -, Pages -Publisher
BMC
DOI: 10.1186/s13007-016-0148-0
Keywords
CRISPR-Cas; Diatom; Genome editing; Urease; Golden Gate; Thalassiosira pseudonana
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Funding
- Natural Environment Research Council (NERC)
- NERC [NE/K004530/1]
- School of Environmental Sciences at University of East Anglia, Norwich
- Natural Environment Research Council [NE/K004530/1, 1316367] Funding Source: researchfish
- NERC [NE/K004530/1] Funding Source: UKRI
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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.
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