Implementation of an alternative genome editing approach via single-strand DNA in the marine microalga Nannochloropsis oceanica

The continuous growth of global population is leading to an increasing demand of food, energy and bio-commodities. In this context, photosynthetic microorganisms as microalgae are considered a valuable complementary source of biomass. Among them, the model organism Nannochloropsis oceanica stands out as valuable candidate for the sustainable production of lipids for industrial purposes. Nevertheless, large-scale cultivation of microalgae is limited due to the high cost of biomass production, low growth and poor productivity, making the derived products not competitive on the market. In this regard, interesting traits for productivity and research can be enhanced through the application of gene editing techniques, based on targeted dsDNA breaks induced by RNA-programmable CRISPR/Cas endonucleases. The consequent DNA repair pathways triggered by the damage can then be exploited to edit genes of interest. However, among these pathways, overall in microalgae, nonhomologous end-joining can generate unexpected insertions and deletions, whereas homologous recombination often requires the use of additional selection markers and occurs with such inefficiency that makes its routine application currently unfeasible. Recently, an emerging alternative strategy, showing a considerably higher efficiency, was developed in the green microalga Chlamydomonas reinhardtii. This technique is based on the introduction of precise and predictable gene edits in vivo, using single-stranded oligodeoxynucleotides (ssODNs). In this thesis the same approach was tested in N. oceanica. I chose the vde gene, that encodes the Violaxanthin de-epoxidase (VDE) as biological target to assess its efficiency. VDE is involved in the xanthophyll cycle, where it converts the carotenoid violaxanthin into zeaxanthin, which then allows the activation of the photoprotection mechanism called non-photochemical quenching (NPQ), that dissipates the excess energy as heat to protect photosynthesis from the consequences of excess light. The deletion of the vde gene is therefore easily measurable as failure in the activation of NPQ. I designed different ssODNs to insert stop codons within the vde gene. After transformation via electroporation I set up a preliminary screening of the transformants to measure the ability to activate NPQ using an Imaging-PAM Fluorometer. I observed that 13% of the transformants were not able to activate NPQ, suggesting a successful disruption of the target vde gene, further confirmed by colony PCR. This confirmed that the ssODN construct is effectively able to physically integrate at the CRISPR/Cas12a-induced double strand breaks. However, the efficiency was 0,3% in this organism, highlighting that at present this is not enough to enable a routine application.