The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Editing events are characterized by loss of heterozygosity and by the occurrence of large deletions of up to ~2.7-kb centred on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-FOA and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyl transferase activity in knockout strains. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.

中文翻译：

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Cas9产生的Tricornutumumumcorn的营养缺陷型的特征是小缺失和大缺失，可以通过基于质粒的基因来补充

模型硅藻Phaeodactylum tricornutum是合成生物学应用的有吸引力的候选者。Tricornutum的营养缺陷型菌株的开发可以为常用的抗生素抗性基因提供替代选择标记。在这里，使用CRISPR / Cas9，我们显示了尿嘧啶，组氨酸和色氨酸生物合成途径中基因的成功编辑。编辑事件的特征在于杂合性的丧失和以编辑位点为中心的最大〜2.7kb的大缺失的出现。固化Cas9编辑质粒后，需要尿嘧啶和组氨酸的表型可以与完整基因的基于质粒的拷贝互补。尿嘧啶营养缺陷型在补充有5-FOA和尿嘧啶的培养基上的生长导致互补质粒的丢失，为质粒固化提供了一种简便的方法，在菌株工程和CRISPR编辑中具有潜在的应用前景。尿嘧啶营养缺陷型的代谢组学表征揭示了细胞乳清蛋白浓度的变化与敲除菌株中乳清酸磷酸核糖基转移酶活性的部分或全部丧失相一致。我们的结果扩大了P. tricornutum营养缺陷型菌株，并证明营养缺陷型互补标记为传统使用的抗生素选择标记提供了可行的替代方法。基于质粒的营养缺陷型标记应扩大基因组工程应用的范围，并为生物工程化的角果疟原虫菌株的生物污染提供一种手段。