Multiplexed Knockouts in the Model Diatom Phaeodactylum by Episomal Delivery of a Selectable Cas9
Multiplexed Knockouts in the Model Diatom Phaeodactylum by Episomal Delivery of a Selectable Cas9
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DOI：
10.3389/fmicb.2020.00005
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发表时间：
2020-01-28
影响因子：
5.2
通讯作者：
Allen, Andrew E.
中科院分区：
文献类型：
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作者：
Moosburner, Mark Andrew;Gholami, Pardis;Allen, Andrew E.
海洋硅藻是一种真核微藻，在海洋中发挥着重要的生态和地球化学作用。它们还具有作为开发生物技术和工业目标的生物平台的巨大潜力。为了解决这两种研究模式，需要先进的分子和遗传工具。我们在这里提出了将CRISPR-Cas9引入模型硅藻三角褐指藻细胞的新的和改进的方法，以及对具有先前未知表型的突变细胞系进行基因分型的简化方案。首先，通过2A肽将Cas9转录融合到选择性标记物来优化细菌缀合以递送Cas9。开发了使用阴性和阳性选择的附加体克隆策略以简化CRISPR-附加体组装。接下来，开发了利用手动测序策展、TIDE测序分析和T7内切核酸酶测定的细胞系挑选和基因分型策略，以缩短产生突变体所需的时间。在这种新的实验管道之后，分别以48%和25%的诱变效率产生单基因和双基因敲除细胞系。最后，使用粒子轰击转化方法开发了通过CRISPR-Cas9靶向进行精确基因插入的方案。总的来说，本文提出的新型Cas9附加体设计和改进的基因分型方法允许快速且容易地对褐指藻突变体细胞系进行基因分型和分离（少于3周），而不依赖于已知的表型来筛选突变体。
Marine diatoms are eukaryotic microalgae that play significant ecological and biogeochemical roles in oceans. They also have significant potential as organismal platforms for exploitation to address biotechnological and industrial goals. In order to address both modes of research, sophisticated molecular and genetic tools are required. We presented here new and improved methodologies for introducing CRISPR-Cas9 to the model diatom Phaeodactylum tricornutum cells and a streamlined protocol for genotyping mutant cell lines with previously unknown phenotypes. First, bacterial-conjugation was optimized for the delivery of Cas9 by transcriptionally fusing Cas9 to a selectable marker by the 2A peptide. An episome cloning strategy using both negative and positive selection was developed to streamline CRISPR-episome assembly. Next, cell line picking and genotyping strategies, that utilize manual sequencing curation, TIDE sequencing analysis, and a T7 endonuclease assay, were developed to shorten the time required to generate mutants. Following this new experimental pipeline, both single-gene and two-gene knockout cell lines were generated at mutagenesis efficiencies of 48% and 25%, respectively. Lastly, a protocol for precise gene insertions via CRISPR-Cas9 targeting was developed using particle-bombardment transformation methods. Overall, the novel Cas9 episome design and improved genotyping methods presented here allow for quick and easy genotyping and isolation of Phaeodactylum mutant cell lines (less than 3 weeks) without relying on a known phenotype to screen for mutants.