Direct in situ protein tagging in Chlamydomonas reinhardtii utilizing TIM, a method for CRISPR/Cas9-based targeted insertional mutagenesis.
Direct in situ protein tagging in Chlamydomonas reinhardtii utilizing TIM, a method for CRISPR/Cas9-based targeted insertional mutagenesis.
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DOI：
10.1371/journal.pone.0278972
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
Witman, George B.
中科院分区：
文献类型：
--
作者：
Hou, Yuqing;Cheng, Xi;Witman, George B.
莱茵衣藻是许多细胞过程研究的重要模式生物，蛋白质标记是这些研究中越来越不可或缺的工具。为了规避传统方法在创建标记细胞系中的缺点，其涉及用随机插入基因组的外源DNA构建体转化野生型或空突变细胞系，我们开发了原位标记内源基因的策略。该策略利用TIM，这是一种基于CRISPR/Cas9的方法，用于在C中进行靶向插入诱变。莱因哈德氏菌我们已经在两个基因上测试了该策略：LF 5/CDKL 5，缺乏该基因会导致长鞭毛表型，以及Cre09.g416350/NAP 1 L1，该基因以前在C.莱因哈德氏菌我们成功地用血凝素（HA）标签标记了野生型LF 5的C-末端，效率为7.4%。测序证实这些菌株被正确编辑。Western blotting证实HA标记的LF 5表达，免疫荧光显微镜显示LF 5-HA正常定位。这些菌株具有正常长度的鞭毛，并且呈现野生型。我们成功地用mNeonGreen-3xFLAG标记Cre09.g416350的N-末端，效率为9%。测序结果表明，这些菌株中的标签区与预期的一致。Western印迹证实了这些菌株中预期大小的标记蛋白的表达，这些菌株似乎具有正常的细胞大小、生长速率和游泳速度。这是C.已经原位编辑了莱茵衣藻内源基因以表达野生型标记蛋白。这种有效、高效、方便的TIM标记策略有望成为研究C.莱因哈德氏菌
Chlamydomonas reinhardtii is an important model organism for the study of many cellular processes, and protein tagging is an increasingly indispensable tool for these studies. To circumvent the disadvantages of conventional approaches in creating a tagged cell line, which involve transforming either a wild-type or null-mutant cell line with an exogenous DNA construct that inserts randomly into the genome, we developed a strategy to tag the endogenous gene in situ. The strategy utilizes TIM, a CRISPR/Cas9-based method for targeted insertional mutagenesis in C. reinhardtii. We have tested the strategy on two genes: LF5/CDKL5, lack of which causes a long-flagella phenotype, and Cre09.g416350/NAP1L1, which has not been studied previously in C. reinhardtii. We successfully tagged the C-terminus of wild-type LF5 with the hemagglutinin (HA) tag with an efficiency of 7.4%. Sequencing confirmed that these strains are correctly edited. Western blotting confirmed the expression of HA-tagged LF5, and immunofluorescence microscopy showed that LF5-HA is localized normally. These strains have normal length flagella and appear wild type. We successfully tagged the N-terminus of Cre09.g416350 with mNeonGreen-3xFLAG with an efficiency of 9%. Sequencing showed that the tag region in these strains is as expected. Western blotting confirmed the expression of tagged protein of the expected size in these strains, which appeared to have normal cell size, growth rate, and swimming speed. This is the first time that C. reinhardtii endogenous genes have been edited in situ to express a wild-type tagged protein. This effective, efficient, and convenient TIM-tagging strategy promises to be a useful tool for the study of nuclear genes, including essential genes, in C. reinhardtii.
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