Genome editing with removable TALEN vectors harboring a yeast centromere and autonomous replication sequence in oleaginous microalga.

Algal lipids are expected to become a basis for sustainable fuels because of the highly efficient lipid production by photosynthesis accompanied by carbon dioxide assimilation. Molecular breeding of microalgae has been studied to improve algal lipid production, but the resultant gene-modified algae containing transgenes are rarely used for outdoor culture because the use of genetically modified organisms (GMOs) is strictly restricted under biocontainment regulations. Recently, it was reported that plasmids containing yeast centromere and autonomous replication sequence (CEN/ARS) behaved as episomes in Nannochloropsis species. We previously reported that the Platinum TALEN (PtTALEN) system exhibited high activity in Nannochloropsis oceanica. Therefore, we attempted to develop a genome editing system in which the expression vectors for PtTALEN can be removed from host cells after introduction of mutations. Using all-in-one PtTALEN plasmids containing CEN/ARS, targeted mutations and removal of all-in-one vectors were observed in N. oceanica, suggesting that our all-in-one PtTALEN vectors enable the construction of mutated N. oceanica without any transgenes. This system will be a feasible method for constructing non-GMO high-performance algae.

Efficient and multiplexable genome editing using Platinum TALENs in oleaginous microalga, Nannochloropsis oceanica NIES-2145.

Algae accumulate large amounts of lipids produced by photosynthesis, and these lipids are expected to be utilized as feedstocks for sustainable new energies, known as biodiesels. Nannochloropsis species are eukaryotic microalgae that produce high levels of lipids. However, since the production costs of algal biodiesels are higher than those of fossil fuels, the improved productivity of algal lipids by molecular breeding of algae is required for practical use. In the present study, we developed a highly efficient genome-editing system involving Platinum transcription activator-like effector nucleases (TALENs) in Nannochloropsis oceanica. Platinum TALENs codon-optimized for N. oceanica were synthesized, and their DNA-binding activity was confirmed by single-strand annealing assays in human HEK293T cells. All-in-one expression vectors for Platinum TALEN targeting the nitrate reductase gene, NoNR, and acyltransferase gene, LPAT1, were transfected into Nannochloropsis species. The introduction of each Platinum TALEN revealed high genome-editing efficiency with no detectable off-target mutations at the candidate sites in N. oceanica. By simultaneously introducing TALENs targeting two genes, we obtained double mutant strains. The loss-of-function phenotype of NoNR was also confirmed. These findings will provide an essential technology for molecular breeding in Nannochloropsis species.