EAGER: A Transient System for Cassava Genome Editing

木薯（Manihot esculenta Crantz）是非洲仅次于玉米的第二大作物。东非是世界上最大的木薯生产国，约占世界产量的60%，通常种植在边际土地上。数以百万计的人依赖木薯作为他们的主要热量来源，它在发展中国家的粮食安全中发挥着关键作用。它也是非洲小农的一个重要收入来源，而小农往往是妇女。许多不同的木薯品种适应当地的环境条件，并具有独特的块茎特性，在非洲各地种植。农民往往更喜欢当地品种，而不是不易受疾病和虫害影响的“改良”品种，这大大限制了非洲的木薯生产。因此，重要的是要发展遗传方法，以改善当地品种以及作为国家育种计划一部分的优良品种。 这个EAGER项目将开发一种创新的、快速的策略来修改木薯基因组，除了所需的突变之外，不会留下任何“足迹”。 这一策略有许多潜在的应用，并将补充传统的育种和转基因方法用于木薯改良。 此外，由于木薯双生病毒是包括近300种病毒物种的菜豆病毒属的成员，所产生的信息和资源也可以为其他菜豆病毒如何适应作为瞬时基因编辑系统的病毒载体提供新的见解，该项目将开发一种新的方法，使用病毒载体表达靶向的核酸酶将位点特异性修饰引入木薯基因组。该系统将使用基于木薯双生病毒的病毒载体来表达大范围核酸酶，该大范围核酸酶旨在将突变引入编码八氢番茄红素去饱和酶（PDS）的木薯基因中，PDS是类胡萝卜素生物合成途径中的第一种酶。PDS突变体的漂白表型将用于筛选由瞬时基因编辑系统引起的基因敲除并确定其功效。将测试几个参数对系统效率的影响，例如接种方法、接种部位、病毒移动和增强感染的木薯序列。实验还将表征后代植物中突变PDS序列以及病毒和大范围核酸酶序列的存在。这些研究将表明接种木薯的营养繁殖是否增加了检测位点特异性突变的效率，以及后代植物是否不保留病毒或大范围核酸酶DNA。

Cassava (Manihot esculenta Crantz) is the second most important crop in Africa after maize. East Africa is the world's largest producer of cassava, accounting for ~60% of world production, where it is often grown on marginal lands. Millions of people depend on cassava as their major source of calories, and it plays a key role in food security for the developing world. It is also an important source of income for small farmers, who are often women in Africa. Many different cassava cultivars that are adapted to local environmental conditions and have unique tuber properties are grown across Africa. Farmers often prefer local varieties instead of "improved" cultivars that are less susceptible to disease and pests, which greatly limit cassava production in Africa. Thus, it is important to develop genetic approaches to improve local varieties as well as elite cultivars that are part of national breeding programs. This EAGER project will develop an innovative, rapid strategy for modifying the cassava genome that leaves no "footprint" except for the desired mutation. This strategy has many potential applications and would complement conventional breeding and transgenic approaches used today for cassava improvement. In addition, since cassava geminiviruses are members of the begomovirus genus that includes nearly 300 viral species, the information and resources generated could also provide new insight into how other begomoviruses can be adapted as viral vectors for transient gene editing systems, potentially extending the technology to many important crop species in addition to cassava.This project will develop a new approach using viral vectors to express targeted nucleases to introduce site-specific modifications into the cassava genome. The system will use a viral vector based on a cassava geminivirus to express a meganuclease designed to introduce mutations into the cassava gene encoding phytoene desaturase (PDS), the first enzyme in the carotenoid biosynthetic pathway. The bleached phenotype of a PDS mutant will be used to screen for gene knockouts caused by the transient gene editing system and establish its efficacy. The impact of several parameters, e.g. the inoculation method, inoculation site, virus movement and cassava sequences that enhance infection, on the efficiency of the system will be tested. Experiments will also characterize progeny plants for the presence of mutant PDS sequences as well as for viral and meganuclease sequences. These studies will indicate if vegetative propagation of the inoculated cassava increases the efficiency of detecting a site-specific mutation and if progeny plants do not retain viral or meganuclease DNA.