A “CRISPY” tale on microalgal genome editing: featuring Nannochloropsis oceanica IMET1

Microalgae are unicellular microbes that can grow under autotrophic conditions to produce valuable compounds such as omega-3 fatty acids, vitamins and lipids. Moreover, these organisms are reported to have faster growth rates and improved photosynthetic efficiencies compared to land plants. These characters make them a promising candidate for sustainable production of green chemicals.

The lipids that are produced by microalgae can be used for producing biofuels. Oleaginous microalgae such as Nannochloropsis oceanica, is found to accumulate more than 50% of its dry weight with lipids under specific conditions. As microalgae cultivation does not compete with land areas that are used for food and feed crop production, this could be a better alternative to the biofuels that are produced from land plants.

The high lipid accumulation in N. oceanica is reported to be triggered under nitrogen stress. However, under these conditions, the growth rate is reduced, and it affects the overall productivity of lipid production. This has been a major bottleneck in commercializing the biofuels produced from microalgae.

Among various strategies to address this bottleneck, metabolic engineering of N. oceanica to improve the lipid productivities has been the focus of this study. Efficient genetic tools that can generate knockouts and knock-ins are crucial for performing metabolic engineering in N. oceanica.

The first part of this study is focussed on developing a genome editing toolbox for microalgae N. oceanica. To this end, we initially reviewed all the advancements made in developing a CRISPR-Cas based genetic toolbox in microalgae. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) proteins are widely used across organisms to specifically target the genomic DNA and engineer the target region with high precision. We found that an approach based on transforming Cas ribonucleoproteins directly into model microalgae Chlamydomonas reinhardtii was an efficient and straightforward approach to generate mutants. The target sites were knocked in the mutants by insertion of indels in C. reinhardtii. We adapted this approach with modifications to generate precise mutants by homology directed repair in N. oceanica. Additionally, we compared multiple variants of Cas proteins (SpCas9, FnCas12a, LbCas12a and AsCas12a) by this approach and identified that FnCas12a as the best Cas variant for engineering N. oceanica.

The presence of an antibiotic resistance cassette in the mutants generated by this approach was a bottleneck as it limited the subsequent engineering of the mutants due to limited availability of antibiotics for N. oceanica. Thus, we further developed this approach to establish a strategy that produced marker-less and scarless mutants of N. oceanica using Cas RNPs.   

The generation of marker-less mutants using RNPs was laborious and had two rounds of transformation. To address this bottleneck, we developed an episomal plasmid based Cas12a system for N. oceanica. The ability of Cas12a to process the CRISPR array was exploited to generate multiplexed mutants in a single transformation. We demonstrated simultaneous targeting at up to three different target sites using this system. This development facilitates high throughput genome engineering in N. oceanica.

Furthermore, a CRISPR interference system for target gene downregulation was developed by expressing catalytically inactive versions of spCas9 and fnCas12a from the episomal plasmid. SpCas9 was found to be a better variant for gene downregulation compared to fnCas12a.

Next, we attempted to perform prime editing in N. oceanica to introduce precise edits at the target site rather than introducing uncontrolled indels. However, this system was not completely functional and will require further optimizations to work efficiently in N. oceanica.

In addition to the targeted genome engineering, to identify novel target genes associated with lipid accumulation, an insertional mutagenesis library of N. oceanica was developed. High lipid producing mutants were selected by five rounds of fluorescence-activated cell sorting and the location of integration cassette was mapped in the host genome. We identified that the disruption of putative APETALA2-like transcription factor gene increased the neutral lipid content by 40% without any growth impairment.

In addition to lipids, omega-3 fatty acid EPA (Eicosapentaenoic acid) is a valuable metabolite that can be produced by N. oceanica. EPA has various health benefits and has been conventionally obtained from fish oils. Accumulating EPA in the triglycerides were found to be a stable source of EPA for human consumption. To this end, an EPA specific lysophosphatidic acid acyltransferase from plant Brassica napus was expressed in N. oceanica. We observed that under nitrogen starved conditions, EPA was improved by 38% in the triglycerides. Thus, BnLPAAT can be a potential candidate in developing tailored triglyceride molecules with high EPA content.

Finally, we critically review the current trends in engineering microalgae to produce valuable compounds and provide novel strategies that could be implemented to improve microalgal productivities. We propose that engineering the upstream part of the metabolism is crucial to improve the microalgal productivities and develop commercially feasible green chemicals from microalgae. The comprehensive genetic toolbox developed in this study with novel insights into the metabolism will be an asset to pursue these ambitious engineering strategies in N. oceanica.