Nuclease-resistant DNA nanostructures for high precision plant genome engineering

背景：CRISPR-Cas9带来了精确基因组工程的可能性，但由于背景随机整合的障碍仍然存在。本项目将应用最先进的纳米技术来提高crispr介导的植物物种靶向基因组修饰的效率。如果我们要满足粮食和能源生产可持续增长的需求，应对气候变化、耕地有限和自然资源压力增加的挑战，就必须有针对性地改进作物。目的：纳米技术允许设计DNA结构，通过形成二级和高阶复合物来抵抗核酸酶的消化。这些结构缺乏自由的双末端，不会成为介导随机转基因整合的末端连接途径的底物，从而促进整合到同源定向途径中。这将丰富新作物品种产生中hr介导的整合事件。新颖性：这种创新和高度变革性的方法将开发新的基于纳米技术的方法，在植物基因组工程中具有潜在的高收益，并应用于其他真核生物物种。及时性：该项目结合了两个领先团队在CRISPR-Cas9技术和新型纳米分子DNA结构方面的互补专业知识，在物理和生命科学领域具有广泛的应用。这些新技术的结合为基因组工程和合成生物学提供了新的可能性。实验方法：将植物细胞转化为DNA无末端转基因的环状DNA，以提供连续的环状结构。更复杂的高度填充的DNA结构也将通过使用短链DNA片段来形成，这赋予了对核酸酶攻击的高抵抗力。该转基因将以内源性卟啉原氧化酶基因为靶点，使其对除草剂butafenicil产生抗性。生物转化植物将进一步分析以确认基因靶向。一个精确的机制理解整合途径将提供通过反向遗传方法，以确定定点插入的遗传决定因素。

Background: CRISPR-Cas9 has brought the possibility precise genome engineering, yet barriers remain due to background random integration. This project will apply state of the art nanotechnology to improve the efficiency of CRISPR-mediated targeted genome modification of plant species. Directed improvement of crops is essential if we are to meet the demand for sustainable increased food and energy production against the challenges of climate change, limited land for cultivation and increased pressure on natural resources. Objectives: Nanotechnology allows the design of DNA structures that are resistant to nuclease digestion, through the formation of secondary and higher order complexes. These structures, lacking free duplex ends, will not be substrates for end-joining pathways that mediate random transgene integration, thereby promoting integration into homology directed pathways. This will enrich HR-mediated integration events in the generation of novel crop varieties. Novelty: This innovative and highly transformative approach will develop novel nanotechnology-based methodologies with potential high gain for genome engineering in plants, with application to other eukaryotic species.Timeliness: The project combines complimentary expertise of two leading groups working with CRISPR-Cas9 technologies and novel nanomolecular DNA structures with wide ranging applications across physical and life sciences. Bringing together these new technologies provides new possibilities for genome engineering and synthetic biology.Experimental approach: Plant cells will be transformed with a DNA end-free transgenes of looped DNA to provide a contiguous ring structure. More complex highly packed DNA structures will also be formed through the use of staple DNA moieties allows that confer high resistance to nuclease attack. The transgene will target the endogenous PROTOPORPHYRINOGEN OXIDASE gene to confer resistance to the herbicide butafenicil. Biolistic transformed plants will be further analysed to confirm gene targeting. A precise mechanistic understanding of integration pathway will be provided through a reverse genetic approach to identify the genetic determinants of site-directed insertion.