BTT EAGER: Plant genome editing and engineering via novel nanotechnology-based systems

国家和全球都需要开发产量更高的粮食、饲料、纤维和生物能源相关产品的农作物。与此同时，有必要开发对非生物和生物胁迫具有更高耐受性并减少对环境影响的作物。基因工程和基因组编辑是农作物研究和改良的重要工具。然而，目前的基因工程/编辑系统不能有效地工作，或者在某些情况下根本不能在几个作物物种/品种中工作。无法对某些种类的植物进行基因工程改造，阻碍了相关作物的进展。该项目旨在开发一种利用超小、可生物降解的合成颗粒（纳米颗粒）对植物进行基因工程/编辑的新系统。这样一个系统的开发将使作物基因组学研究和具有增强性状和性能的作物的生产能够更快地取得进展。此外，通过本研究产生的知识可用于增强其他基因组编辑应用。基因组工程/编辑系统是推进植物功能基因组学和表观基因组学研究以及基于基因组学的作物改良工作的关键工具。当前植物基因组工程/编辑系统的主要局限性——如基因型依赖性、低效率和可变效率，以及/或无法完成无整合的种系编辑，严重阻碍了这些技术在作物基因组学研究和遗传改良应用中的高效、广泛利用。为了克服这些限制，并提高植物基因组编辑的效率和广度，正在开发一种基于纳米粒子的系统，用于将生物工程分子机制引入能够再生种系编辑植物的靶细胞。该研究项目的具体目的是：证明纳米颗粒介导的Cas9/sgRNA核糖蛋白（RNP）复合物以不依赖基因型的方式进入靶细胞，并成功编辑植物细胞/组织中的靶DNA序列，以及基因组编辑植物的再生。新型可生物降解的纳米颗粒系统（纳米胶囊和复合体）被用于将RNPs输送到最佳的植物细胞/组织靶标中，以产生基因组编辑的植物细胞和组织，从中可以恢复种系编辑的植物。将进行显微镜、视觉和分子分析，以确定基因组编辑的成功、位置、频率和遗传。这里学到的知识不仅可以应用于植物系统，也可以应用于非植物编辑应用。开发一种高效的、不依赖基因型的纳米颗粒植物基因编辑系统将对基于基因组工程的功能基因组学研究和作物改良工作产生重大的积极影响。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。

There is a national and global need to develop crop plants that yield greater food, feed, fiber and bioenergy related products. At the same time there is a need to develop crops with an increased tolerance to abiotic and biotic stress and reduced impacts on the environment. Genetic engineering and genome editing are important tools used for the study and improvement of crop plants. However, current genetic engineering/editing systems do not work efficiently, or in some cases at all in several crop species/varieties. The inability to genetically engineer certain species of plants, hinders the progress that can be made with related crops. This project is aimed at developing a new system to genetically engineer/edit plants using ultra-small, biodegradable, synthetic particles (nanoparticles). Development of such a system would enable more rapid advancement in crop genomics research and production of crops with enhanced traits and performance. In addition, the knowledge generated through this research may be utilized in the enhancement of other genome editing applications. Genome engineering/editing systems are critical tools for the advancement of plant functional genomics and epigenomics research, and for genomics-based crop improvement efforts. Major limitations in current plant genome engineering/editing systems - such as genotype dependence, low and variable efficiencies, and/or inability to accomplish integration-free, germline editing have significantly impeded efficient, widespread utilization of these technologies for crop genomics research and genetic improvement applications. To overcome these limitations, and to increase the efficiency and breadth of plant genome editing a nanoparticle-based systems for introducing bioengineering molecular machinery into target cells capable of regenerating germline edited plants is being developed. The specific aim of the research project is: to demonstrate nanoparticle-mediated delivery of Cas9/sgRNA ribonuleoprotein (RNP) complexes into target cells in a genotype-independent manner, and successful editing of target DNA sequences in plant cells/tissues, as well as regeneration of genome-edited plants. Novel, biodegradeable nanoparticle-based systems (nanocapsule and polyplex) are used to deliver RNPs into optimal plant cell/tissue targets to generate genome-edited plant cells and tissues from which germline edited plants can be recovered. Microscopic, visual and molecular assays will be conducted to determine genome editing success, location, frequency and inheritance. What is learned here can be applied not only to plant systems, but also in non-plant editing applications. Development of an efficient, genotype-independent nanoparticle-based plant gene editing system would have significant positive impact on genome engineering-based functional genomics research and crop improvement efforts.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.