随着基因组编辑技术的日渐成熟，植物基因定点敲除变得简单易行,但是通过同源重组途径实现精确的定点替换和插入等基因靶向修饰技术在植物中仍是急待攻克的难题。本研究拟以CRISPR/Cas系统为重心，通过：1）优化Cas9缺刻酶制造DNA单链缺口；2）利用双生病毒转化体系提高供体模板DNA表达量；3）定点敲除ZmKu70阻断NHEJ途径等多途径入手进行综合调控——以ZmIPK1、ZmUbi1、ZmDst1和ZmEPSPS为研究对象，在玉米HiII和综31两个品种的原生质体、未成熟胚、愈伤和植物中进行系统分析。研究将建立起玉米高效基因组靶向修饰技术体系，推进植物基因组编辑技术的进步，为玉米基因功能解析和分子定向育种提供技术支撑；同时通过基因定点替换ZmEPSPS基因关键核苷酸创制生物安全的抗除草剂玉米优异种质资源，为解决转基因玉米的生物安全问题提供新思路，推进基因组编辑育种技术在玉米生产应用的步伐。

Recent advances in CRISPR/Cas system make it possible to precisely alter DNA sequences in living cells, providing unprecedented control over a plant’s genetic material. However, most of these mutations are created by non-homologous end joining (NHEJ)-mediated gene knockout, homologous recombination (HR)-mediated gene replacement or insertion is still a big challenge in plant. In this project, we will optimize CRISPR/Cas system by testing Cas9 nickases for their ability to stimulate homologous recombination, developing germinivirus as vectors for gene targeting in maize and knockouting ZmKu70 , which can block NHEJ pathway and therefore activate HR pathway. We will test these factors in protoplast, immature embryo, callus and plants from two maize varieties of HiII and zong31. We will further apply these optimized technologies for creating glyphosate-resistant plants through gene replacement of key nucleotides of ZmEPSPS gene. This study will not only establish new genome editing technology for maize gene function analysis and molecular breeding but also provide the insight into the ability to introduce new genetic variation into maize by genome editing breeding technology.