Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems

[Image: see text] Microalgae can produce industrially relevant metabolites using atmospheric CO(2) and sunlight as carbon and energy sources, respectively. Developing molecular tools for high-throughput genome engineering could accelerate the generation of tailored strains with improved traits. To this end, we developed a genome editing strategy based on Cas12a ribonucleoproteins (RNPs) and homology-directed repair (HDR) to generate scarless and markerless mutants of the microalga Nannochloropsis oceanica. We also developed an episomal plasmid-based Cas12a system for efficiently introducing indels at the target site. Additionally, we exploited the ability of Cas12a to process an associated CRISPR array to perform multiplexed genome engineering. We efficiently targeted three sites in the host genome in a single transformation, thereby making a major step toward high-throughput genome engineering in microalgae. Furthermore, a CRISPR interference (CRISPRi) tool based on Cas9 and Cas12a was developed for effective downregulation of target genes. We observed up to 85% reduction in the transcript levels upon performing CRISPRi with dCas9 in N. oceanica. Overall, these developments substantially accelerate genome engineering efforts in N. oceanica and potentially provide a general toolbox for improving other microalgal strains.