Maize 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 the complex genome and genetic architecture. Here we integrated multiplexed CRISPR/Cas9-based high-throughput targeted mutagenesis with genetic mapping and genomic approaches to successfully knock-out 743 candidate genes corresponding to agronomic and nutritional traits. After low-cost barcode-based deep sequencing, 412 edited sequences covering 118 genes were precisely identified from individuals showing clear phenotypic changes. This mutant profile was similar to the ones identified in human cell lines and predictable. An unexpectedly frequent homology-directed repair through endogenous templates was observed and likely caused by the spatial contact between distinct chromosomes. Through several case studies on validation and interpretation of gene function, this targeted mutagenesis library suggested that the integration of forward- and reverse- genetics 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.

玉米是世界上最重要的农作物之一。然而，由于复杂的基因组和遗传结构，很少有农学上重要的玉米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图谱和基因组方法相集成，以成功剔除743个与农艺和营养性状相对应的候选基因。经过低成本的基于条形码的深度测序后，从显示明显表型变化的个体中精确鉴定了涵盖118个基因的412个编辑序列。该突变体概况与在人类细胞系中鉴定出的和可预测的相似。观察到通过内源性模板出乎意料的频繁的同源性指导的修复，可能是由于不同染色体之间的空间接触所致。通过一些关于基因功能验证和解释的案例研究，该靶向诱变文库表明，正向和反向遗传学的整合有望对具有复杂基因组的农作物快速验证重要的农艺基因。除了特定的发现，这项研究还指导了植物中高通量CRISPR实验的进一步优化。