CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum

Marianne Nymark; Amit Kumar Sharma; Marthe C G Hafskjold; Torfinn Sparstad; Atle M Bones; Per Winge

doi:10.21769/BioProtoc.2442

CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum

Bio Protoc. 2017 Aug 5;7(15):e2442. doi: 10.21769/BioProtoc.2442.

Authors

Marianne Nymark¹, Amit Kumar Sharma¹, Marthe C G Hafskjold¹, Torfinn Sparstad¹, Atle M Bones¹, Per Winge¹

Affiliation

¹ Department of Biology, Norwegian University of Science and Technology, Trondheim, Norway.

Abstract

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.

Keywords: Biolistic transformation; CRISPR/Cas9 technology; Diatoms; HRM analyses; Phaeodactylum tricornutum.