EAGER: Lambda-Red-enhanced homologous recombination in plants

EAGER：Lambda-Red 增强植物中的同源重组

• 批准号: 1940829
• 负责人: Jose Alonso
• 金额: $ 30万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1940829&HistoricalAwards=false
• 关键词: EAGER; Lambda; Red; enhanced; homologous

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.

在生物体的基因组中编辑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，而且还改变了如何处理诸如人类疾病，粮食安全，生物燃料生产等关键社会问题的解决方案。目前在动植物系统中使用的主要基因组编辑技术要求在需要更改或编辑的精确位置引入基因组中的断裂。尽管高效，但目前的基因组编辑程序仍然艰苦，并且没有由于基因组中不受欢迎的断裂而没有任何风险。尽管不依赖基因组中染色体断裂引入的高效基因组编辑方法已经存在，但仅证明它们在细菌系统中起作用。利用当前对无DNA基因组编辑方法的工作以及其要求是什么，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。除了为植物开发新的无DNA基因组编辑方法的好处外，产生的知识还将为动物系统的类似发展提供信息。该项目将为研究人员和本科生提供理想的培训平台，以亲身体验如何将基本生物学知识转化为潜在的变革性新技术。为了进一步通知公众并激励年轻科学家，该项目将在当地博物馆主持有关植物生物学和遗传学原理的研讨会。 该项目的主要目标是开发真核细胞的基于Lambda-RED的同源重组（HR）系统。这种系统的实施不仅可以为当前的基因组编辑方法提供替代方案，还可以提供重要的优势，例如消除在基因组中引起潜在危险的DSDNA断裂的需求，非常短的序列同源性要求，高效效率以及从单核段变化到大型插入物质和大型插入物质和dellets的潜力。重要的是，已经对Lambda-Red系统背后的分子机制进行了彻底的研究，从而为需要满足的要求提供了关键的线索，以便测试真核系统中Lambda-Red介导的HR的可行性。这些要求包括：1）积极复制基因组，2）可以用作修复模板的线性DNA片段，以及3）针对细胞核的主动重组酶。为了满足这些要求，将在植物细胞中与病毒衍生的复制子一起表达密码子优化的，核定核的重新组合酶，该酶可以作为修复模板DNA的来源。也将表达一个高度优化的核定核核酸酶，以线性化病毒复制子来生成所需的线性修复模板。如果成功的话，这项工作将为农业及其他方面的利益开辟新的精确基因组操纵手段。该奖项反映了NSF的法定任务，并被认为是值得通过基金会的知识分子和更广泛影响的评估评估来支持的。