Format

Send to

Choose Destination
Plant Methods. 2016 Nov 24;12:49. eCollection 2016.

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana.

Author information

1
School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ UK.
2
The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH UK.

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 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.

KEYWORDS:

CRISPR-Cas; Diatom; Genome editing; Golden Gate; Thalassiosira pseudonana; Urease

Supplemental Content

Full text links

Icon for BioMed Central Icon for PubMed Central
Loading ...
Support Center