EAGER: Development of an RNA editing tool for probing the function of plant mitochondrial genes and proteins

本项目旨在开发使能技术，用于研究植物线粒体的基本基因功能。在植物细胞中，就像在动物细胞中一样，线粒体被称为细胞动力源，产生基本活动所需的能量。线粒体包含它们自己的基因组，已知植物的基因组比动物的基因组更复杂、更大；然而，植物基因组是如何产生特异性RNA和蛋白质产物的还不清楚。此外，研究线粒体基因的遗传工具也很缺乏。因此，这个项目产生的工具可能会彻底改变分析和潜在地设计线粒体基因及其产物的能力。除了这种科学影响外，该项目还将通过为本科生提供实践研究培训，为他们成为未来STEM劳动力的成员做好准备，从而产生有益的教育成果。该项目旨在通过提供一种允许编辑特定核苷酸的线粒体转录本的技术，解决缺乏遗传工具来探测线粒体基因功能的问题。基因组编辑领域的CRISPR革命依赖于核酸酶（Cas蛋白）的使用，它通过在入侵的噬菌体dna中产生双链断裂，在细菌免疫中发挥作用。最近，失去核酸内切酶活性但保留其与核酸结合亲和力的“死亡”Cas蛋白已被用于设计作用于DNA或RNA的核单碱基编辑器。这个项目的目的是为线粒体RNA开发一个类似的工具。这一研究成果将为植物线粒体生物学研究提供一种新技术。这将为了解线粒体基因的功能、基因调控序列和蛋白质提供前所未有的机会，因为它可以精确地改变编码线粒体蛋白质的核基因，而这些基因无法通过突变来实现。这种方法也可以提供一种基因工程植物线粒体功能的方法。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。

This project aims to develop enabling technology for studying essential gene functions in plant mitochondria. In plant cells, like in animals, mitochondria are known as the cellular powerhouses, generating the energy needed for essential activities. Mitochondria contain their own genomes, which are known to be more complex and much larger in plants than in animals; yet, how plant genomes work to produce characteristic RNA and protein products is not well understood. Moreover, genetic tools for studying genes in mitochondria are lacking. Therefore, the tools to be generated by this project could revolutionize the ability to analyze and potentially engineer mitochondrial genes and their products. In addition to this scientific impact, the project will have beneficial educational outcomes by providing undergraduate students with hands-on research training to prepare them to become members of the future STEM workforce.This project aims to resolve the lack of genetic tools to probe mitochondrial gene functions by providing a technology that will allow editing of mitochondrial transcripts at certain nucleotides. The CRISPR revolution in genome editing relies on the use of nucleases, Cas proteins, which act in bacterial immunity by generating double-stranded breaks in invading phage DNAs. Recently, "dead" Cas proteins that have lost their endonuclease activity but retained their binding affinity for nucleic acids have been used to engineer nuclear single base editors acting on either DNA or RNA. This project aims to develop a similar tool for mitochondrial RNA. The impact of this work will provide a novel technology for the field of plant mitochondrial biology. It will open up unprecedented opportunities to understand the function of mitochondrial genes, gene regulatory sequences, and proteins by making precise changes that cannot be made by mutating nuclear genes that encode mitochondrial proteins. This methodology could also provide a way to genetically engineer plant mitochondrial function.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.