Utilization of microalgae has been hampered by limited tools for creating loss-of-function mutants. Furthermore, modified strains for deployment into the field must be free of antibiotic resistance genes and face fewer regulatory hurdles if they are transgene free. The oleaginous microalga, Nannochloropsis oceanica CCMP1779, is an emerging model for microalgal lipid metabolism. We present a one-vector episomal CRISPR/Cas9 system for N. oceanica that enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal DNA. The vector utilizes a bidirectional promoter to produce both Cas9 and a ribozyme flanked sgRNA. This system efficiently generates targeted mutations, and allows the loss of episomal DNA after the removal of selection pressure, resulting in marker-free nontransgenic engineered lines. To test this system, we disrupted the nitrate reductase gene (NR) and subsequently removed the CRISPR episome to generate nontransgenic marker-free nitrate reductase knockout lines (NR-KO).

中文翻译：

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在卵生微藻，Nannochloropsis oceanica CCMP1779中由游离体CRISPR系统进行的非转基因无标记基因破坏

微藻的利用已经受到用于产生功能丧失突变体的有限工具的阻碍。此外，用于野外部署的改良菌株必须不含抗生素抗性基因，并且如果不含转基因，则面临的监管障碍也较少。含油微藻Nannochloropsis oceanica CCMP1779是微藻脂质代谢的新兴模型。我们提出了一种用于大洋念珠菌的单载体游离型CRISPR / Cas9系统，该系统能够生成无标记的突变株。酿酒酵母的CEN / ARS6区载体中包含了β-环糊精，以促进其作为环状染色体外DNA的维持。该载体利用双向启动子来产生Cas9和侧接sgRNA的核酶。该系统有效产生靶向突变，并在去除选择压力后允许游离DNA丢失，从而形成无标记的非转基因工程系。为了测试该系统，我们破坏了硝酸盐还原酶基因（NR），随后去除了CRISPR附加体以产生非转基因的无硝酸盐还原酶敲除品系（NR-KO）。