Simultaneous knockout of multiple LHCF genes using single sgRNAs and engineering of a high-fidelity Cas9 for precise genome editing in marine algae.

The CRISPR/Cas9 system is an RNA-guided sequence-specific genome editing tool, which has been adopted for single or multiple gene editing in a wide range of organisms. When working with gene families with functional redundancy, knocking out multiple genes within the same family may be required to generate a phenotype. In this study, we tested the possibility of exploiting the known tolerance of Cas9 for mismatches between the single-guide RNA (sgRNA) and target site to simultaneously introduce indels in multiple homologous genes in the marine diatom Phaeodactylum tricornutum.

Expression of DNA repair genes in arctic char (Salvelinus alpinus) from Bjørnøya in the Norwegian Arctic.

High levels of organochlorines (OCs) have been measured in arctic char (Salvelinus alpinus) from Lake Ellasjøen on Bjørnøya, Norway (74.30°N, 19.0°E).

Assessment of oxidative stress response genes in exposed to oil contamination - Polyphenol oxidase as a biomarker.

Mangrove plants, which inhabit and form sensitive ecosystems in the intertidal zones of tropical and subtropical coastlines, though vulnerable to petroleum pollution, still maintain their growth under oil contamination. To elucidate the molecular response of mangrove plants to crude oil-sediment mixture, seeds of were planted and grown on 0, 2.5, 5.

Transgene-free genome editing in marine algae by bacterial conjugation - comparison with biolistic CRISPR/Cas9 transformation.

The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box.

CRISPR/Cas9 Gene Editing in the Marine Diatom .

The establishment of the CRISPR/Cas9 technology in diatoms ( Hopes , 2016 ; Nymark , 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 , 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 cells and 3) a high resolution melting based PCR assay to screen for CRISPR/Cas9 induced mutations.