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

该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，就像在动物中一样，线粒体被称为细胞动力源，产生必要活动所需的能量。线粒体包含它们自己的基因组，众所周知，植物的基因组比动物的更复杂，也更大；然而，植物基因组是如何产生特有的RNA和蛋白质产物的还不是很清楚。此外，目前还缺乏研究线粒体基因的遗传工具。因此，该项目将产生的工具可能会彻底改变分析线粒体基因及其产品的能力，并有可能对其进行工程设计。除了这种科学影响，该项目还将通过为本科生提供动手研究培训，为他们成为未来的STEM工作人员做好准备，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某些核苷酸的线粒体转录本的技术，解决缺乏探测线粒体基因功能的遗传工具的问题。CRISPR在基因组编辑方面的革命依赖于核酸酶的使用，这种蛋白质通过在入侵的噬菌体DNA中产生双链断裂而在细菌免疫中发挥作用。最近，“死亡”的CaS蛋白失去了其内切酶活性，但仍保持了与核酸的结合亲和力，已被用于设计作用于DNA或RNA的核单碱基编辑程序。该项目旨在开发一种类似的线粒体RNA工具。这项工作的影响将为植物线粒体生物学领域提供一种新的技术。它将通过进行编码线粒体蛋白质的核基因突变无法实现的精确改变，为了解线粒体基因、基因调控序列和蛋白质的功能提供前所未有的机会。这种方法还可以提供一种对植物线粒体功能进行基因工程的方法。这一奖项反映了NSF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 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.