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

Abstract

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.

Engineering microalgae as the next-generation food

“…In C. reinhardtii, ZFNs were reported to edit photoreceptor genes via homologous recombination [34]. Naduthodi and coworkers developed a genome editing strategy based on CRISPR/Cas12a to generate a Nannochloropsis oceanica mutant [41]. Besides the development in gene editing technology, the most challenging task is to create high specificity and unexpected target effects.…”

New trends in biotechnological applications of photosynthetic microorganisms

“…sequence-specific recombinant nucleases including zincfinger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN) (Patel et al, 2019;Shin et al, 2016). Since 2014, CRISPR-based genome editing techniques have been used to manipulate the genetic material of some freshwater and marine microalga (Wenzhi et al, 2014;Nymark et al, 2016;Naduthodi et al, 2021). However, there are still many concerns regarding the introduction of Genetically Modified Organisms from the laboratory to the natural environment, mainly due to possibilities of unpredicted gene flow in the natural habitat (Fayyaz et al, 2020).…”

Biochemistry and Biotechnology of Lipid Accumulation in the Microalga Nannochloropsis oceanica

“…oceanica lines at best. The recent development of effective genome editing and transgene expression systems may contribute to the future metabolic engineering efforts. ,,,− For example, a novel chimeric gene expression system using the nucleolus as a genomic safe harbor has been recently established for strong and consistent transgene expression in N. oceanica .…”

“…oceanica strains have been sequenced, including CCMP1779 and IMET1, and additional transcriptomic and proteomic resources for many Nannochloropsis species are available (e.g., Nannochloropsis Design and Synthesis, NanDeSyn, , accessed August 2022). − Moreover, genetic toolboxes for manipulating gene expression in N. oceanica have been successfully developed, including gene stacking for multiple gene overexpression and clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing techniques for precise genome editing ,− (for a review, see ref ). The high lipid and LC-PUFA content along with recent advances in genetic engineering, therefore, promote strong interests in developing N.…”

“…2 Therefore, microalgae hold the promise of a sustainable resource for lipidbased biofuel production as a result of their high lipid accumulation ability and no competition with agricultural crops for arable land. 2 Some microalgal species also produce health-promoting long-chain polyunsaturated fatty acids (LC-PUFAs), such as eicosapentaenoic acid (EPA, C20:5Δ 5,8,11,14,17 ) and docosahexaenoic acid (DHA, C22:6Δ 4,7,10,13,16,19 ). 3,4 EPA and DHA are essential ω-3 fatty acids that have important roles in neuronal and retinal development, immune system, cardiovascular health, and cognitive function and have been widely used in nutritional supplements.…”