Journal
BIO-PROTOCOL
Volume 7, Issue 15, Pages -Publisher
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.2442
Keywords
CRISPR/Cas9 technology; Diatoms; Phaeodactylum tricornutum; Biolistic transformation; HRM analyses
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Funding
- Gordon and Betty Moore Foundation [GBMF 4966]
- NTNU enabling technologies program
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The establishment of the CRISPR/Cas9 technology in diatoms (Hopes et al., 2016; Nymark et al., 2016) enables a simple, inexpensive and effective way of introducing targeted alterations in the genomic DNA of this highly important group of eukaryotic phytoplankton. Diatoms are of interest as model microorganisms in a variety of areas ranging from oceanography to materials science, in nano- and environmental biotechnology, and are presently being investigated as a source of renewable carbon-neutral fuel and chemicals. Here we present a detailed protocol of how to perform CRISPR/Cas9 gene editing of the marine diatom Phaeodactylum tricornutum, including: 1) insertion of guide RNA target site in the diatom optimized CRISPR/Cas9 vector (pKS diaCas9-sgRNA), 2) biolistic transformation for introduction of the pKS diaCas9-sgRNA plasmid to P. tricornutum cells and 3) a high resolution melting based PCR assay to screen for CRISPR/Cas9 induced mutations.
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