EAGER: Lambda-Red-enhanced homologous recombination in plants

编辑生物体基因组信息的能力不仅改变了科学家解决基本生物学问题的方式，也改变了解决人类疾病、粮食安全、生物燃料生产等关键社会问题的方法。目前在植物和动物系统中使用的主要基因组编辑技术需要在基因组中需要改变或编辑的精确位置引入断裂。虽然效率很高，但目前的基因组编辑程序仍然很费力，而且也不是没有风险，其中包括不希望出现的基因组断裂。尽管不依赖于在基因组中引入染色体断裂的高效基因组编辑方法已经存在，但它们只被证明在细菌系统中有效。利用目前对dna无断裂基因组编辑方法如何工作及其要求的理解，该项目旨在修改现有细菌系统的组成部分，并使其适应植物细胞的功能。除了开发一种新的无dna断裂的植物基因组编辑方法的好处之外，所产生的知识还将为动物系统的类似开发提供信息。该项目将为研究人员和本科生提供一个理想的培训平台，亲身体验如何将基础生物学知识转化为潜在的变革性新技术。为了进一步向公众宣传和激励年轻科学家，该项目将在当地一家博物馆举办植物生物学和遗传学原理讲习班。本项目的主要目标是为真核细胞开发基于lambda - red的同源重组（HR）系统。该系统的实现不仅为当前的基因组编辑方法提供了一种替代方案，而且还可能提供重要的优势，例如消除了在基因组中诱导潜在危险的dsDNA断裂的需要，非常短的序列同源性要求，高效率，以及从单核苷酸改变到大插入和缺失产生大阵列修饰的潜力。重要的是，我们已经深入研究了Lambda-Red系统背后的分子机制，从而为验证Lambda-Red介导的HR在真核系统中的可行性提供了关键线索。这些要求包括：1)一个积极复制的基因组，2)一个可以作为修复模板的线性DNA片段，以及3)针对细胞核的活跃重组酶。为了满足这些需求，密码子优化的、核定位的重组酶将在植物细胞中与病毒衍生的复制子一起表达，该复制子可以作为修复模板DNA的来源。一个密码子优化的，核定位的巨核酶也将被表达，以线性化病毒复制子，以产生所需的线性修复模板。这项工作如果成功，将为农业和其他领域的精确基因组操作的新手段打开大门。该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知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The ability to edit the information in an organism's genome is transforming not only the way scientists address basic biological questions, but also how solutions to deal with critical societal problems such as human diseases, food security, biofuel production, etc. are sought. The predominant genome editing technologies currently in use in plants and animal systems require the introduction of breaks in the genome at the precise locations where the changes or edits are wanted. Although highly efficient, current genome editing procedures are still laborious and not free of risks due, among other things, to undesired breaks in the genome. Although high-efficiency genome editing approaches that do not rely on the introduction of chromosomal breaks in the genome already exist, they have only been proven to work in bacterial systems. Using the current understanding of how DNA-break-free genome editing approaches work and what their requirements are, this project aims to modify components of an existing bacterial system and adapt them to function in plant cells. In addition to the benefits of developing a new DNA-break-free genome editing approach for plants, the generated knowledge would inform similar developments in animal systems. This project will provide an ideal training platform for researchers and undergraduate students to experience first-hand how basic biological knowledge can be translated into potentially transformative new technologies. To further inform the public and motivate young scientists, the project will lead workshops at a local museum on principles of plant biology and genetics. The main goal of this project is to develop a Lambda-Red-based homologous recombination (HR) system for eukaryotic cells. The implementation of such system would not only provide an alternative to the current genome editing approaches, but may also offer important advantages, such as the elimination of the need to induce potentially dangerous dsDNA breaks in the genome, very short sequence homology requirements, high efficiency, and the potential to generate large arrays of modifications, from single nucleotide changes to large insertions and deletions. Importantly, the molecular mechanisms behind the Lambda-Red system have been thoroughly investigated, thus providing critical clues on the requirements that need to be fulfilled in order to test the feasibility of Lambda-Red-mediated HR in a eukaryotic system. These requirements include: 1) an actively replicating genome, 2) a linear DNA fragment that can serve as the repair template, and 3) active recombineering enzymes targeted to the nucleus of the cell. To fulfill these requirements, codon-optimized, nuclear-localized recombineering enzymes will be expressed in plant cells together with a virus-derived replicon that can serve as the source for repair template DNA. A codon-optimized, nuclear-localized meganuclease will be also expressed to linearize the viral replicons to generate the required linear repair template. The work, if successful, will open doors to new means of precise genome manipulation for the benefits of agriculture and beyond.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.