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

Microalgae can produce
industrially relevant metabolites using
atmospheric CO2 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.