EAGER: Lambda-Red-enhanced homologous recombination in plants

编辑生物体基因组中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，而且改变了如何寻求解决人类疾病，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处引入断裂。尽管高效，但目前的基因组编辑程序仍然是费力的，并且由于基因组中不期望的断裂等原因而没有风险。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，但它们仅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。除了为植物开发一种新的无DNA断裂的基因组编辑方法的好处外，所产生的知识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一个理想的培训平台，让他们亲身体验基本生物知识如何转化为具有潜在变革性的新技术。为了进一步向公众宣传和激励年轻科学家，该项目将在当地博物馆举办关于植物生物学和遗传学原理的讲习班。 该项目的主要目标是开发一种基于λ-Red的真核细胞同源重组（HR）系统。这样的系统的实施将不仅提供当前基因组编辑方法的替代方案，而且还可以提供重要的优点，例如消除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，以及产生从单核苷酸变化到大插入和缺失的大修饰阵列的潜力。重要的是，λ-Red系统背后的分子机制已经得到了彻底的研究，从而为测试λ-Red介导的HR在真核系统中的可行性所需满足的要求提供了关键线索。这些要求包括：1）活跃复制的基因组，2）可用作修复模板的线性DNA片段，和3）靶向细胞核的活性重组工程酶。为了满足这些要求，密码子优化的、核定位的重组工程酶将与病毒衍生的复制子一起在植物细胞中表达，所述病毒衍生的复制子可以用作修复模板DNA的来源。还将表达密码子优化的、核定位的大范围核酸酶以使病毒复制子线性化，从而产生所需的线性修复模板。这项工作，如果成功的话，将打开大门，以精确的基因组操纵的新手段，为农业和超越的benefits.This奖项反映了NSF的法定使命，并已被认为是值得的支持，通过评估使用基金会的知识价值和更广泛的影响审查标准。

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.