High-throughput genotyping of green algal mutants reveals random distribution of mutagenic insertion sites and endonucleolytic cleavage of transforming DNA

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.

针对单细胞光合真核生物的高通量遗传筛选平台，将成为植物生物学研究工具箱中一项极具变革意义的工具。本文报道了ChlaMmeSeq（基于MmeI的衣藻插入位点测序法，Chlamydomonas MmeI-based insertion site Sequencing），一款可同时对真核单细胞绿藻莱茵衣藻（Chlamydomonas reinhardtii）中的数万个诱变插入位点进行定位的研究工具。我们首先通过对单个插入位点的深度表征验证了ChlaMmeSeq的性能。随后我们将ChlaMmeSeq应用于突变体库，成功定位到11478个插入位点，覆盖了已注释蛋白质编码基因的39%。我们观察到插入位点的分布模式与随机分布几乎无差异，这表明几乎所有基因的突变体均可高效获取。本研究数据揭示了序列特异性核酸内切酶活性可切割转化DNA，并据此提出了一个简单模型，用以解释有时环绕插入位点的、尚未被充分阐明的外源序列的来源。ChlaMmeSeq具有定量可重复性，可用于混合富集筛选以及构建带索引的突变体库。此外，ChlaMmeSeq可以前所未有的规模对衣藻筛选得到的阳性克隆进行基因分型，为全面鉴定参与光合作用、藻类脂质代谢、藻类碳浓缩机制、趋光性、纤毛生物发生与功能以及其他以莱茵衣藻为主要模式系统的生物学过程的基因提供了可能。