PlantSynBio: Development and application of a versatile CRISPR platform for simultaneous gene editing and transcriptional regulation in plants

植物合成生物学是工程原理与植物生物学相结合的新兴领域。为了加快合成生物学的设计-测试-建造-学习循环，需要在植物中开发新的工具。近年来，用于改变基因序列的基因编辑工具和用于改变基因表达的基因调控工具都在植物中得到了开发。然而，植物研究界还没有能够同时实现基因编辑和基因调控的工具。为了填补这一重大技术空白，将开发一种能够同时进行基因编辑、基因激活和基因抑制的工具箱。这些工具将在大米中得到演示，大米是养活世界三分之一人口的主要作物。示范将包括开发高产黄金大米，这为治疗人类维生素A缺乏症提供了一个有前途的解决方案，以及开发具有高营养价值的抗除草剂原花青素生物强化大米。该项目将激发和放大社区对将新的合成生物学工具应用于下一代代谢工程的兴趣，以培育出高产、更有营养、更抗病、更能适应气候变化的更好的作物。培训未来的科学家，特别是来自代表性不足的少数群体的科学家，是这一研究项目的核心任务。基于CRISPR-CAS的基因组工程工具的发展极大地促进了作物育种和代谢工程的发展。为了释放植物合成生物学和系统生物学的潜力，需要同时操作植物基因组和转录组的创新工具。该项目旨在开发和应用一个简单易用、高效和多功能的CRISPR-CAS9平台，用于同时和正交地进行植物基因编辑、转录激活和抑制。该平台将通过生产具有其他所需性状的并行工程的生物强化水稻来展示。该项目的目标是(1)开发用于植物基因编辑和转录调控的新型CRISPR系统，(2)应用同步基因编辑和激活系统来设计高产黄金水稻，(3)应用同时基因编辑、激活和抑制系统来设计抗除草剂原花青素生物强化水稻。尽管水稻被用于技术开发，但这些工具与其他植物的应用程序兼容。为这个多功能CRISPR-CAS9平台开发的载体将提供给其他研究人员，以便它们可以被植物研究社区广泛采用，用于基础和翻译研究。该项目的更广泛影响包括对马里兰大学学院公园分校、加州大学戴维斯分校和华盛顿霍华德大学的本科生进行培训。该项目还为蒙哥马利·布莱尔高中和埃莉诺·罗斯福高中的学生提供STEM培训。该奖项反映了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。

Plant synthetic biology is an emerging field that combines engineering principles with plant biology. To speed up the design-test-build-learn cycle in synthetic biology, new tools need to be developed in plants. In recent years, gene editing tools for altering gene sequences and gene regulation tools for altering gene expression have both been developed in plants. However, tools that allow for achieving gene editing and gene regulation at the same time are not yet available to the plant research community. To fill in this major technology gap, a toolbox capable of inducing simultaneous gene editing, gene activation and gene repression will be developed. These tools will be demonstrated in rice, a major crop that feeds a third of the world population. Demonstrations will include the development of high-yield Golden Rice, which provides a promising solution for treating vitamin A deficiency in humans, and the development of herbicide-resistant proanthocyanidin-biofortified rice with high nutrition value. This project will ignite and amplify community interest in applying new synthetic biology tools for next-generation metabolic engineering to make better crops that are high-yield, more nutritious, disease-resistant and more resilient in a changing climate. Training future scientists, especially from under-represented minority groups, is a core mission of this research project. The development of CRISPR-Cas based genome engineering tools has greatly accelerated crop breeding and metabolic engineering. To unleash the potential of plant synthetic biology and systems biology, innovative tools are required for simultaneous manipulation of the plant genome and transcriptome. This proposed project aims to develop and apply an easy-to-use, highly efficient and multifunctional CRISPR-Cas9 platform for simultaneous and orthogonal gene editing, transcriptional activation and repression in plants. The platform will be demonstrated by generating biofortified rice with concurrent engineering of other desirable traits. The project aims are to (1) develop novel CRISPR systems for simultaneous gene editing and transcriptional regulation in plants, (2) apply a simultaneous gene editing and activation system to engineer high-yield Golden Rice, and (3) apply a simultaneous gene editing, activation and repression system to engineer herbicide-resistant proanthocyanidin-biofortified rice. Although rice is used for technology development, the tools are compatible with applications in other plants. The vectors developed for this multifunctional CRISPR-Cas9 platform will be made available to other researchers so that they can be widely adopted by the plant research community for basic and translational research. The broader impacts of this project include training for undergraduate students at University of Maryland at College Park, University of California at Davis and Howard University at Washington. The project also provides STEM training to students from Montgomery Blair High School and Eleanor Roosevelt High School.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.