Tanaka Naoto, Mogi Yuko, Fujiwara Takayuki, Yabe Kannosuke, Toyama Yukiho, Higashiyama Tetsuya, Yoshida Yamato
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan.
Department of Gene Function and Phenomics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
J Cell Sci. 2021 Nov 1;134(21). doi: 10.1242/jcs.258948. Epub 2021 Nov 10.
The unicellular alga Cyanidioschyzon merolae has a simple cellular structure; each cell has one nucleus, one mitochondrion, one chloroplast and one peroxisome. This simplicity offers unique advantages for investigating organellar proliferation and the cell cycle. Here, we describe CZON-cutter, an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease for targeted editing of any locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants using fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows us to validate phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner. This article has an associated First Person interview with the first author of the paper.
单细胞藻类梅氏蓝纤维藻具有简单的细胞结构；每个细胞有一个细胞核、一个线粒体、一个叶绿体和一个过氧化物酶体。这种简单性为研究细胞器增殖和细胞周期提供了独特的优势。在这里，我们描述了CZON-cutter，一种经过工程改造的成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关核酸酶9（Cas9）系统，用于同时进行基因组编辑和细胞器可视化。我们构建了一种梅氏蓝纤维藻菌株，该菌株表达一种核定位的Cas9-维纳斯核酸酶，用于对由单向导RNA（sgRNA）定义的任何位点进行靶向编辑。然后，我们成功地编辑了藻类基因组，并使用具有不同激发波长的荧光蛋白报告基因在转化体中对线粒体和过氧化物酶体进行了可视化。活转化体中细胞器的荧光蛋白标记使我们能够验证与细胞器增殖和细胞周期相关的表型，即使被编辑的基因是必需的。结合梅氏蓝纤维藻的特殊生物学特性，CZON-cutter将有助于以高通量方式研究细胞和细胞器分裂。本文配有对该论文第一作者的第一人称访谈。
