High-Throughput CRISPR/Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize
High-Throughput CRISPR/Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize
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高通量 CRISPR/Cas9 诱变简化了玉米性状基因鉴定
DOI：
10.1105/tpc.19.00934
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发表时间：
2020-05-01
期刊：
影响因子：
11.6
通讯作者：
Yan, Jianbing
中科院分区：
文献类型：
--
作者：
Liu, Hai-Jun;Jian, Liumei;Yan, Jianbing
玉米（Zea mays）是世界上最重要的作物之一。然而，由于其复杂的基因组和遗传结构，很少有重要的农艺学基因被克隆并用于性状改良。在这里，我们将基于CRISPR/ cas9的多重高通量靶向诱变与遗传作图和基因组方法相结合，成功靶向了743个与农学和营养相关性状对应的候选基因。通过低成本的条形码深度测序，从个体中精确鉴定出412个编辑序列，覆盖118个基因，显示出明显的表型变化。相关基因编辑事件的概况与在人类细胞系中发现的相似，因此可以使用最初为人类研究设计的现有算法进行预测。我们通过内源性模板观察到意想不到但频繁的同源定向修复，这可能是由不同染色体之间的空间接触引起的。基于几个例子的基因功能表征和解释，我们证明了通过靶向诱变文库整合正向和反向遗传学可以快速验证具有复杂基因组的作物的重要农艺基因。除了具体发现外，本研究还指导了植物高通量CRISPR实验的进一步优化。
Maize (Zea mays) is one of the most important crops in the world. However, few agronomically important maize genes have been cloned and used for trait improvement, due to its complex genome and genetic architecture. Here, we integrated multiplexed CRISPR/Cas9-based high-throughput targeted mutagenesis with genetic mapping and genomic approaches to successfully target 743 candidate genes corresponding to traits relevant for agronomy and nutrition. After low-cost barcode-based deep sequencing, 412 edited sequences covering 118 genes were precisely identified from individuals showing clear phenotypic changes. The profiles of the associated gene-editing events were similar to those identified in human cell lines and consequently are predictable using an existing algorithm originally designed for human studies. We observed unexpected but frequent homology-directed repair through endogenous templates that was likely caused by spatial contact between distinct chromosomes. Based on the characterization and interpretation of gene function from several examples, we demonstrate that the integration of forward and reverse genetics via a targeted mutagenesis library promises rapid validation of important agronomic genes for crops with complex genomes. Beyond specific findings, this study also guides further optimization of high-throughput CRISPR experiments in plants.