High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA
High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA
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DOI：
10.1105/tpc.114.124099
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发表时间：
2014-04-01
期刊：
影响因子：
11.6
通讯作者：
Jonikas, Martin C.
中科院分区：
文献类型：
--
作者：
Zhang, Ru;Patena, Weronika;Jonikas, Martin C.
单细胞光合真核生物的高通量遗传筛选平台将是植物生物学工具箱的变革性补充。在这里，我们提出了ChlaMmeSeq（衣原体MmeI为基础的插入位点测序），成千上万的诱变插入位点的真核单细胞绿色衣藻莱茵衣藻同时映射的工具。我们首先通过深入表征单个插入位点来验证ChlaMmeSeq。然后，我们将ChlaMmeSeq应用于突变体库，并映射了11，478个插入，覆盖了39%的注释蛋白质编码基因。我们观察到，插入分布的方式在很大程度上是难以区分的随机，表明几乎所有的基因突变体可以有效地获得。数据显示，序列特异性内切核酸酶活性切割转化DNA，并使我们能够提出一个简单的模型来解释有时围绕插入位点的人们知之甚少的外源序列的起源。ChlaMmeSeq是定量可重复的，使其能够用于合并富集筛选和产生索引突变体文库。此外，ChlaMmeSeq允许以前所未有的规模对衣原体筛选的命中进行基因分型，为全面鉴定在光合作用、藻类脂质代谢、藻类碳浓缩机制、趋光性、纤毛的生物发生和功能以及C. reinhardtii是一个领先的模型系统。
A high-throughput genetic screening platform in a single-celled photosynthetic eukaryote would be a transformative addition to the plant biology toolbox. Here, we present ChlaMmeSeq (Chlamydomonas MmeI-based insertion site Sequencing), a tool for simultaneous mapping of tens of thousands of mutagenic insertion sites in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. We first validated ChlaMmeSeq by in-depth characterization of individual insertion sites. We then applied ChlaMmeSeq to a mutant pool and mapped 11,478 insertions, covering 39% of annotated protein coding genes. We observe that insertions are distributed in a manner largely indistinguishable from random, indicating that mutants in nearly all genes can be obtained efficiently. The data reveal that sequence-specific endonucleolytic activities cleave the transforming DNA and allow us to propose a simple model to explain the origin of the poorly understood exogenous sequences that sometimes surround insertion sites. ChlaMmeSeq is quantitatively reproducible, enabling its use for pooled enrichment screens and for the generation of indexed mutant libraries. Additionally, ChlaMmeSeq allows genotyping of hits from Chlamydomonas screens on an unprecedented scale, opening the door to comprehensive identification of genes with roles in photosynthesis, algal lipid metabolism, the algal carbon-concentrating mechanism, phototaxis, the biogenesis and function of cilia, and other processes for which C. reinhardtii is a leading model system.