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High-Throughput CRISPR/Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize

文献类型： 外文期刊

作者： Liu, Hai-Jun 1 ; Jian, Liumei 1 ; Xu, Jieting 1 ; Zhang, Qinghua 1 ; Zhang, Maolin 1 ; Jin, Minliang 1 ; Peng, Yong 1 ; Y 1 ;

作者机构： 1.Huazhong Agr Univ, Natl Key Lab Crop Genet Improvement, Wuhan 430070, Peoples R China

2.WIMI Biotechnol Co Ltd, Changzhou 213000, Peoples R China

3.Yunnan Acad Agr Sci, Xishuangbanna Inst Agr Sci, Kunming 650205, Yunnan, Peoples R China

4.Jilin Acad Agr Sci, Agrobiotechnol Inst, Jilin Prov Key Lab Agr Biotechnol, Changchun 130033, Peoples R China

5.Biogle Genome Editing Ctr, Changzhou 213125, Peoples R China

6.Beijing Acad Agr & Forestry Sci, Beijing Key Lab Maize DNA Fingerprinting & Mol Br, Beijing 100097, Peoples R China

7.Cold Spring Harbor Lab, POB 100, Cold Spring Harbor, NY 11724 USA

8.Max Planck Inst Mol Plant Physiol, D-14476 Potsdam, Germany

期刊名称：PLANT CELL （ 影响因子：11.277； 五年影响因子：12.061 ）

ISSN： 1040-4651

年卷期： 2020 年 32 卷 5 期

页码：

收录情况： SCI

摘要： 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.

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