Genetic Tool Development for Nannochloropsis species

Algae are excellent candidates for providing a renewable source of liquid fuels with an overall reduction in carbon emissions compared to conventional petroleum fuels. However, the economic viability of algal biofuel production at industrial scales is limited by the low biomass productivities of natural algal species. Strain development of algal species has lagged behind conventional industrial microbes due to the lack of available tools for genetic manipulation. In this work, we develop tools for DNA delivery, nuclear genome editing via CRISPR-Cas9 technology, and chloroplast expression in Nannochloropsis species. High voltage electroporation and acoustic-based delivery (i.e., sonoporation) methods are investigated for three species of Nannochoropsis: gaditana, oceanica, and salina. Variability in transformation efficiencies are detected among these three species, with N. salina showing the lowest transformation efficiency under electroporation. Reduced transformation efficiencies are observed with the addition of Cas9 to the genome integration construct, which is consistent with the Cas9 toxicity observed in other algal species. The expression of genome-integrated Cas9 in N. oceanica is investigated under different growth and environmental conditions. Lastly, three chloroplast-targeting sequences were fused with a green fluorescent protein to demonstrate chloroplast targeting of genes integrated into the nuclear genome. These transformation, gene editing, and chloroplast expression tools will enable the modification of Nannochloropsis species to improve biomass productivities in support of algal biofuels production as well as other chemical products produced from CO₂ via photosynthesis.