Nuclease-resistant DNA nanostructures for high precision plant genome engineering

用于高精度植物基因组工程的抗核酸酶 DNA 纳米结构

• 批准号: 2271151
• 金额: --
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2271151
• 关键词: Nuclease; resistant; DNA; nanostructures; precision

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.

背景：CRISPR-Cas9为精确的基因组工程带来了可能性，但由于背景随机整合，仍然存在障碍。该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。如果我们要在气候变化、耕地有限和自然资源压力增加的挑战下满足可持续增加粮食和能源生产的需求，定向改良作物至关重要。目的：纳米技术允许通过形成二级和高级复合物来设计耐核酸酶消化的DNA结构。缺乏游离双链体末端的这些结构将不是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性指导的途径中。这将丰富HR介导的整合事件在新的作物品种的产生。新奇：这一创新和高度变革性的方法将开发基于纳米技术的新方法，为植物基因组工程提供潜在的高收益，并应用于其他真核生物物种。时间：该项目结合了两个领先团队的互补专业知识，致力于CRISPR-Cas9技术和新型纳米分子DNA结构，在物理和生命科学中具有广泛的应用。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：植物细胞将被转化为环状DNA的无末端转基因，以提供连续的环状结构。还将通过使用赋予对核酸酶攻击的高抗性的钉合DNA部分来形成更复杂的高度堆积的DNA结构。转基因将靶向内源性原卟啉原氧化酶基因，以赋予对除草剂布他非尼西的抗性。将进一步分析生物射弹转化的植物以确认基因靶向。通过反向遗传学方法鉴定定点插入的遗传决定因素，将提供对整合途径的精确机制理解。