Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest.

中文翻译：

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通过无DNA基因组编辑一步一步生成硅藻Phaeodactylum tricornutum中的多个基因敲除。

最近开发的用于探索和开发微藻代谢潜能的转基因技术存在与将外源DNA传递到细胞中以及随后在基因组内随机位点整合有关的几个缺陷。在这里，我们报告了一种高效的多重基因组编辑方法，它依赖于CRISPR-Cas9核糖核蛋白的生物弹传递以及两个内源性反选择标记PtUMPS和PtAPT的鉴定，在硅藻Phaeodactylum tricornutum中实现了高效的多重基因组编辑方法。首先，我们通过积极选择每个基因的破坏来证明RNP传递的功能。然后，我们通过使用针对这些标记之一和PtAureo1a的RNP生成效率为65％至100％的双基因敲除菌株，说明了这种方法的潜力。编码感光体的基因。最后，我们通过将六种RNP复合物递送到Phaeodactylum细胞中一步创建了三重敲除菌株。这种方法可以很容易地应用于生物技术感兴趣的其他难以转染的生物。