CAREER: Nanoparticle-mediated genome engineering of plants and plastids

植物基因工程是可持续发展努力、药物合成和农业工程的核心。有效的植物基因工程面临的主要瓶颈是生物分子通过坚硬的多层细胞壁进入植物细胞。这一限制限制了可以通过基因工程改造的植物的种类。在这个项目中，将开发纳米颗粒来将遗传物质输送到植物叶绿体中，叶绿体是负责光合作用的含有基因的细胞器。该项目将首先专注于分子生物学工具和纳米颗粒的开发，这些工具和纳米颗粒可以穿过植物细胞壁，并将生物分子运送到植物叶绿体中。接下来，该项目将测试基于纳米颗粒的叶绿体输送工具的效率，并将它们与传统工具进行比较，目标是实现更高的遗传转化效率。该项目将通过将基因组编辑生物分子整合到纳米粒子中来演示叶绿体中的基因组编辑。拟议方法的潜在影响将是实现植物叶绿体的基因工程，从而使叶绿体基因的发现和研究以及叶绿体用于生产有价值的蛋白质产品的潜在用途成为可能。在宣传和教育方面，将举办一个国际虚拟会议，将科学家、政策制定者和公众聚集在一起，讨论科学主题和在媒体上传播文献。将开发一门本科课程，让本科生接触同行评议过程和假设驱动的研究，高中教师将在夏季被带到实验室进行实践研究培训。这些努力将提高公众、大学和学校的科学素养。尽管创造转基因植物很重要，但植物系统，特别是非模式植物物种，仍然很难进行基因改造。植物转化的主要瓶颈是植物转化或基因沉默所需的分子生物学工具的传递：DNA、RNA和蛋白质，跨越植物细胞壁，特别是在以叶绿体为目标的遗传操作研究中。在这个项目中，纳米粒子将被开发和功能化，以不同物种和高效的方式吸附和运送质粒DNA到植物叶绿体。为此，在目标1中，将开发基因组编辑质粒DNA载体并将其输送到模型植物叶片和组织中，以测试基因组编辑效率并确认缺乏转基因整合。这一目标将证实纳米材料进行叶绿体遗传转化的能力，其效率超过传统工具。目标2随后将开发并优化一个基于荧光素酶的质粒报告系统，以便在植物叶绿体中表达荧光素酶，目标是超过生物传递DNA所能达到的转化效率。最后，在目标3中，将根据在目标1和2中获得的知识开发基因组编辑质粒，以针对模式植物和非模式植物中的内源叶绿体基因。通过基因操作叶绿体的能力，特别是在非模式植物物种中，将扩大目前可以研究的植物的范围。植物叶绿体基因组的遗传操作反过来又将独特地使大多数光合作用蛋白编码基因的研究成为可能，特别是在非模式植物中。该项目还将利用叶绿体特有的功能，包括缺乏基因沉默途径，以实现转基因的高效表达。后一种特性使叶绿体成为生物合成生产商品化学品、药物，甚至更大的蛋白质产品，如抗体和生物制品的诱人目标。从正交性上讲，由于拟议的方法本质上是非致病的，这种方法的广泛影响可以使对作物物种中转化的植物组织进行更无缝的监管。该项目还将开展三项努力，以提高科学素养。在第一项工作中，将开发一个国际虚拟会议，将科学家、政策制定者和公众聚集在一起，讨论科学主题和通过媒体传播文学。在第二项努力中，将开发一门本科课程，让本科生接触同行评议过程和假设驱动的研究。在第三项努力中，高中教师将在暑期被带到实验室进行动手研究培训。这些努力将提高公众、大学和学校的科学素养。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。

Genetic engineering of plants is at the core of sustainability efforts, drug synthesis, and agricultural engineering. The predominant bottleneck facing efficient plant genetic engineering is biomolecule delivery into plant cells through the rigid and multi-layered cell wall. This constraint limits the species of plants that can be genetically engineered. In this project, nanoparticles will be developed to deliver genetic material into the plant chloroplast, which is a gene-containing organelle responsible for photosynthesis. The project will first focus on the development of molecular biology tools and nanoparticles that can traverse the plant cell wall and deliver biomolecules into plant chloroplasts. Next, the project will test efficiencies of nanoparticle-based chloroplast delivery tools and compare them with conventional tools, with the goal of achieving higher genetic transformation efficiencies. The project will conclude by incorporating genome-editing biomolecules into nanoparticles to demonstrate genome editing in the chloroplast. The potential impact of the proposed approach will be to enable genetic engineering of the plant chloroplast, thus enabling the discovery and study of chloroplast genes and the potential use of chloroplasts for producing valuable protein products. For outreach and education, an international virtual conference will be developed that brings together scientists, policymakers, and the public to discuss scientific topics and the dissemination of literature in the media. An undergraduate course will be developed to expose undergraduate students to the peer-review process and hypothesis-driven research, and high school teachers will be brought into the laboratory for hands-on research training during the summer. These efforts will increase scientific literacy in the public, universities, and schools.Despite the importance of creating transgenic plants, plant systems, particularly non-model plant species, remain difficult to genetically transform. The dominant bottleneck to plant transformation is the delivery of the molecular biology tools needed for plant transformation or gene silencing: DNA, RNA, and protein, across the plant cell wall, particularly for chloroplast-targeted genetic manipulation studies. In this project, nanoparticles will be developed and functionalized to adsorb and deliver plasmid DNA to plant chloroplasts in a species-independent and high-efficiency manner. To this end, in Objective 1, genome editing plasmid DNA vectors will be developed and delivered to model plant leaves and tissues to test genome editing efficiencies and to confirm a lack of transgene integration. This objective will confirm the ability of nanomaterials for chloroplast genetic transformation with efficiencies that surpass those of conventional tools. Objective 2 will subsequently develop, and codon optimize a luciferase-based plasmid reporter system to enable expression of luciferase in plant chloroplasts, with a goal of exceeding transformation efficiencies achievable with biolistic DNA delivery. Lastly, in Objective 3, a genome editing plasmid will be developed based on knowledge gained in Objectives 1 and 2 to target endogenous chloroplast genes in both model and non-model plant species. The ability to genetically manipulate chloroplasts, particularly in non-model plant species, will broaden the range of plants that can currently be studied. Genetic manipulation of the plant chloroplast genome will in turn uniquely enable the study of genes coding for most photosynthetic proteins, especially in non-model plants. This project will also leverage features unique to the chloroplast including a lack of gene silencing pathways, to enable high efficiency expression of transgenes. This latter feature makes chloroplasts an attractive target for biosynthetic production of commodity chemicals, drugs, and even larger protein products such as antibodies and biologics. Orthogonally, because the proposed approach is non-pathogenic in nature, the broader impact of this approach could enable more seamless regulatory oversight of transformed plant tissues in crop species. This project will also develop three efforts to increase scientific literacy. In the first effort, an international virtual conference will be developed that brings together scientists, policymakers, and the public to discuss scientific topics and the dissemination of literature in the media. In the second effort, an undergraduate course will be developed to expose undergraduate students to the peer-review process and hypothesis-driven research. In the third effort, high school teachers will be brought into the laboratory for hands-on research training during the summer. These efforts will increase scientific literacy in the public, universities, and schools.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.