Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 305-701, Republic of Korea.
Sci Rep. 2020 Dec 17;10(1):22158. doi: 10.1038/s41598-020-78968-x.
Recent technical advances related to the CRISPR/Cas9-based genome editing system have enabled sophisticated genome editing in microalgae. Although the demand for research on genome editing in microalgae has increased over time, methodological research has not been established to date for the delivery of a ribonucleoprotein (Cas9/sgRNA complex) using a cell penetrating peptide into microalgal cell lines. Here, we present a ribonucleoprotein delivery system for Chlamydomonas reinhardtii mediated by the cell penetrating peptide pVEC (LLIILRRRIRKQAHAHSK) which is in a non-covalent form. Using this technically simple method, the ribonucleoprotein was successfully delivered into C. reinhardtii. Gene Maa7 and FKB12 were disrupted, and their distinguishing patterns of Indel mutations were analyzed with the observation of several insertions of sequences not originating from the genome DNA, such as chloroplast DNA, into the expected loci. In addition, the cytotoxicity of Cas9 and the ribonucleoprotein was investigated according to the concentration and time in the algal cells. It was observed that Cas9 alone without the sgRNA induces a more severe cytotoxicity compared to the ribonucleoprotein. Our study will not only contribute to algal cell biology and its genetic engineering for further applications involving various organisms but will also provide a deeper understating of the basic science of the CRISPR/Cas9 system.
最近与基于 CRISPR/Cas9 的基因组编辑系统相关的技术进步使微藻中的复杂基因组编辑成为可能。尽管随着时间的推移，对微藻基因组编辑的研究需求不断增加，但迄今为止，尚未建立用于将核糖核蛋白（Cas9/sgRNA 复合物）递送至微藻细胞系的穿透肽的方法研究。在这里，我们提出了一种使用穿透肽 pVEC（LLIILRRRIRKQAHAHSK）介导的用于莱茵衣藻的核糖核蛋白递送系统，该系统处于非共价形式。使用这种技术简单的方法，成功地将核糖核蛋白递送至莱茵衣藻中。基因 Maa7 和 FKB12 被破坏，并分析了它们的 Indel 突变的特征模式，观察到一些源自叶绿体 DNA 而不是基因组 DNA 的序列插入到预期的基因座中。此外，还根据藻类细胞中的浓度和时间研究了 Cas9 和核糖核蛋白的细胞毒性。结果表明，与核糖核蛋白相比，单独的 Cas9 而没有 sgRNA 会引起更严重的细胞毒性。我们的研究不仅将为藻类细胞生物学及其遗传工程提供进一步应用于涉及各种生物体的贡献，而且还将为 CRISPR/Cas9 系统的基础科学提供更深入的理解。
