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

该项目将开发一种新的方法来促进和加速在作物中鉴定成功的同源重组(HR)产物。这种方法是基于“基因组编辑”方面的进展，这是一项革命性的技术，承诺能够在目前遗传资源有限的基因组中引入遗传改进或新功能。在需要用另一个DNA序列替换内源DNA片段的植物中的应用是可能的，但由于HR率非常低，效率非常低。该项目开发的技术旨在克服这一瓶颈，目的是通过直接修改一系列感兴趣的基因，极大地促进基因功能的测试，从而促进生物生物学的发展。这些进展将有助于提高对基因组-物候组关系的理解。例如，这可以加速培育对病原体的抵抗力更强的植物，或者提高作物产量。该项目将进行实验，以进一步开发、测试、验证和改进这项技术及其应用，首先测试系统的组件，然后将所学应用于单个和多个目标。这项工作将在木薯和小米以及开花植物模型中测试该系统。该项目的更广泛影响包括广泛传播改良作物和其他物种的方法，以及通过与长期且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通过使用转录激活的基于报告的方法来促进和加速鉴定人力资源成功产品的技术。该方法将插入修复模板(包含所需的基因组编辑和可选择的标记)与快速找到所需的、罕见的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。这项工作将以木薯、拟南芥和狗尾草为主要分析物种，因为在这些物种中的示范将支持在优子叶和单子叶物种中的广泛应用，包括大多数作物。该项目要解决的问题包括：1)是否可以通过专注于产品的选择来提高人力资源的效率；2)是否可以使用基于人力资源的方法来成功地标记非编码RNA，如microRNA和tasiRNA前体；以及3)是否有可能开发高效的高通量、基于文库的敲入过程。该项目将为关键的功能基因组工具(HR产品的改进选择)开发一种方法，以实现对关于不同生物体中基因功能的假设的直接测试。项目成员将接受广泛的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培训。在这个项目中产生的所有资源都将可供使用。具体地说，构建物将被存放在非营利性全球质粒库中，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地设计SURREFIRE的定制组件，用于他们的有机体研究(S)。这一奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.