EDGE: High Efficiency Identification of Products of Homologous Recombination in Plants as a Tool to Test Gene Function

EDGE：高效鉴定植物同源重组产物作为测试基因功能的工具

• 批准号: 1827761
• 负责人: Blake Meyers
• 金额: $ 100万
• 依托单位: Donald Danforth Plant Science Center
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1827761&HistoricalAwards=false
• 关键词: EDGE; Efficiency; Identification; Products; Homologous

This project will develop a new method to facilitate and accelerate the identification of successful products of homologous recombination (HR) in crop plants. This method is based on advances in 'genome editing', a revolutionary technology that promises the ability to introduce genetic improvements or novel functionality to genomes where genetic resources are currently limited. Applications in plants that require replacing an endogenous piece of DNA with another DNA sequence are possible but are very inefficient due to very low rates of HR. The technology developed in this project is designed to overcome this bottleneck, with the aim to advance organismal biology by greatly facilitating the testing of gene function through direct modification of a broad range of genes of interest. Such advances will contribute to improvements in the understanding of the genomes-to-phenomes relationship. This could, for example, accelerate the development of plants with improved resistance to pathogens, or enhanced crop yields. The project will carry out experiments to further develop, test, validate and improve this technology and its application, first testing the components of the system, then applying what is learned to single and multiple targets. The work will test the system in cassava and foxtail millet, as well as in model flowering plants. Broader impacts of the project include the wide dissemination of the method for the improvement of crop plants and other species, plus the training of students via integration with long-running and successful undergraduate internship program. The project will focus specifically on techniques to facilitate and accelerate the identification of successful products of HR by reporter-based methods using transcriptional activation. The approach combines insertion of a repair template (containing the desired genome edit and a selectable marker) with methods to rapidly find a desirable, rare HR event - the proverbial "needle in a haystack". Data from cassava demonstrates that this is an effective method for developing molecular and genomic tools for orphan crops and those lacking robust genomic resources. The work will be done in cassava, Arabidopsis, and Setaria as the primary species for analysis, because a demonstration in these species would support broad utility in eudicot and monocot species, including most crops. The questions to be addressed by this project include: 1) whether the efficiency of HR can be improved by focusing on selection of the products; 2) whether HR-based approaches be used to successfully tag non-coding RNAs such as microRNA and tasiRNA precursors; and, 3) whether it is possible to develop efficient processes for high-throughput, library-based knock-ins. The project will develop an approach for a key functional genomic tool (improved selection of products of HR) to enable direct tests of hypotheses about gene function in diverse organisms. Project members will be trained broadly in plant biology and genomics, gene function and functional genomics, and computational methods. All resources generated in this project will be made available for use. Specifically, constructs will be deposited in nonprofit global plasmid repositories and web-based tools will be designed to enable users unfamiliar with genome editing to successfully design custom components of SureFire for application to their organism(s) of study.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

该项目将开发一种新的方法，以促进和加速作物同源重组（HR）成功产物的鉴定。这种方法是基于“基因组编辑”的进步，这是一种革命性的技术，有望将遗传改良或新功能引入目前遗传资源有限的基因组。在植物中的应用，需要更换一个内源性的DNA片段与另一个DNA序列是可能的，但由于非常低的HR率是非常低效的。在这个项目中开发的技术旨在克服这一瓶颈，其目的是推进生物体生物学大大促进基因功能的测试，通过直接修改广泛的基因的利益。这些进展将有助于改善对基因组与表型组关系的理解。例如，这可以加速植物的发展，提高对病原体的抵抗力，或提高作物产量。该项目将进行实验，以进一步开发、测试、验证和改进这一技术及其应用，首先测试系统的组成部分，然后将学到的知识应用于单个和多个目标。这项工作将在木薯和谷子以及模型开花植物中测试该系统。该项目的更广泛影响包括广泛传播作物和其他物种改良方法，以及通过与长期运行和成功的本科生实习计划相结合对学生进行培训。该项目将特别侧重于技术，以促进和加速识别成功的产品，人力资源的基于标记的方法，使用转录激活。该方法将插入修复模板（包含所需的基因组编辑和选择标记）与快速找到所需的罕见HR事件的方法相结合-谚语中的“大海捞针”。来自木薯的数据表明，这是一种为孤儿作物和缺乏强大基因组资源的作物开发分子和基因组工具的有效方法。这项工作将在木薯、拟南芥和狗尾草中进行，作为分析的主要物种，因为在这些物种中的示范将支持在真双子叶植物和单子叶植物物种中的广泛应用，包括大多数作物。本项目要解决的问题包括：1）是否可以通过专注于产物的选择来提高HR的效率; 2）基于HR的方法是否可以成功地标记非编码RNA，如microRNA和tasiRNA前体;以及，3）是否可能开发用于高通量的有效方法，该项目将开发一种关键功能基因组工具（改进HR产物的选择）的方法，以便能够直接测试关于不同生物体中基因功能的假设。项目成员将接受植物生物学和基因组学、基因功能和功能基因组学以及计算方法方面的广泛培训。该项目产生的所有资源都将提供使用。具体而言，构建体将存放在非营利的全球质粒库中，基于网络的工具将被设计成使不熟悉基因组编辑的用户能够成功设计SureFire的定制组件，以应用于他们的研究生物体。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。