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.

中文翻译：

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在含油微藻Nannochloropsis oceanica NIES-2145中使用铂TALENs进行高效且可复用的基因组编辑。

藻类会积聚大量通过光合作用产生的脂质，这些脂质有望用作可持续的新能源的原料，被称为生物柴油。拟南芥属物种是产生高水平脂质的真核微藻类。然而，由于藻类生物柴油的生产成本比化石燃料的生产成本高，因此实际应用需要通过藻类分子育种来提高藻类脂质的生产率。在本研究中，我们开发了一种高效的基因组编辑系统，该系统涉及Nannochloropsis oceanica中的Platinum转录激活因子样效应子核酸酶（TALEN）。针对大洋猪笼草优化的铂金TALENs密码子合成，并通过单链退火测定法在人HEK293T细胞中证实了其DNA结合活性。将针对硝酸盐还原酶基因NoNR和酰基转移酶基因LPAT1的TALEN铂的多合一表达载体转染到Nannochloropsis物种中。每个白金TALEN的引入显示了高的基因组编辑效率，在海洋猪笼草的候选位点没有可检测到的脱靶突变。通过同时引入靶向两个基因的TALEN，我们获得了双突变株。还证实了NoNR的功能丧失表型。这些发现将为纳米拟南芥物种的分子育种提供必要的技术。