TRTech-PGR: NSF BSF - Recombining Allelic Diversity via Genome Editing for Dissecting Complex Plant Traits

全球可持续发展努力有赖于植物育种技术的改进，特别是与人类消费相关的作物，如谷类作物。驯化植物的野生近亲拥有许多基因，这些基因使植物能够变得健壮，抵御压力。然而，由于识别植物抗逆性基因所需的实验吞吐量较低，我们很难识别这些基因并将它们的功能分配给植物抗逆性。限制作物遗传弹性测试的一个主要瓶颈是将生物分子送入植物细胞的能力，植物细胞壁为引入植物遗传图谱所需的分子生物学工具提供了基本上不可逾越的障碍。在这个项目中，将开发纳米颗粒来将遗传物质输送到植物细胞中，重点是谷类作物大麦。这些纳米粒子将被化学功能化，以将基因组编辑货物运送到植物中，这将使探测特定的植物基因与植物抗逆性相关。该项目将寻求确定哪些基因使谷类作物对环境压力具有健壮的抵抗力，并利用这一知识了解与全球粮食供应相关的其他作物的抗逆性。上述研究还将支持人数不足的本科生作为纳米材料合成和表征的研究实习生，并将发展一个关于纳米材料科学的学生讲习班。研究植物的基因型与表型的关系对于鉴定植物的生物和非生物胁迫基因是至关重要的。具体地说，作物野生近亲拥有赋予作物适应气候变化相关特征的基因。CRISPR基因组编辑技术的出现极大地促进了通过功能基因组学来映射基因与表型之间的关系。然而，植物基因组编辑的效率限制了这种方法在功能基因组学中的应用。在这个项目中，纳米颗粒将被开发用于以CRISPR为基础的DNA质粒和Cas9-gRNA核糖核蛋白复合体的物理化学吸附。这些纳米颗粒-生物分子货物复合体将在模式植物和谷类作物大麦中进行传递和基因组编辑效率测试。以非整合的方式传递CRISPR质粒或通过Cas9-gRNA复合体传递实现无DNA基因组编辑的能力将通过避免转基因整合和转基因分离的需要而使植物性状的定位更快和更准确。此外，基于纳米颗粒的DNA或蛋白质输送的非生物性质可能使其他相关作物的遗传操作和功能基因组学变得容易，而不是目前工作中测试的作物。除了上述努力外，这项提案还将支持对奇卡诺人和美洲原住民科学促进会本科生的培训，以及开发一个研讨会，向来自不同背景的学生教授纳米材料科学。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。

Global sustainability efforts rely on improvements in plant breeding technology particularly for crops of relevance for human consumption such as cereal crops. Wild relatives of domesticated plants harbor many of the genes that enable plants to become robust against stress. However, our ability to identify these genes and allocate their function to plant stress resilience has been difficult due to the low throughput of experiments needed to identify plant resilience genes. A main bottleneck limiting the throughput of testing crop genetic resilience is the ability to deliver biological molecules into plant cells, where the plant cell wall presents a largely impenetrable barrier for the introduction of the molecular biology tools needed for plant genetic mapping. In this project, nanoparticles will be developed to deliver genetic material into plant cells, with a focus on cereal crop barley. These nanoparticles will be chemically functionalized to deliver genome editing cargoes into plants, which will enable probing of specific plant genes in their relevance for plant stress tolerance. The project will seek to identify what genes enable cereal crops to be robust against environmental stress, and to leverage this knowledge to learn about stress tolerance in other crops of relevance for the global food supply. The research above will also support underrepresented undergraduate students as research trainees for the synthesis and characterization of nanomaterials and will develop a student workshop on nanomaterials science. The ability to study genotype to phenotype relationships in plants is critical for identification of plant biotic and abiotic stress genes. Specifically, crop wild relatives harbor genes that confer traits relevant for crop adaptation to climate change. Mapping genotype to phenotype relationships through functional genomics has benefitted greatly from the emergence of CRISPR genome editing technologies. However, the efficiency of genome editing in plants limits the utility of this method for functional genomics. In this project, nanoparticles will be developed for the physiochemical adsorption of CRISPR-based DNA plasmids and, separately, Cas9-gRNA ribonucleoprotein complexes. These nanoparticle-biomolecule cargo complexes will be tested for delivery and genome editing efficacy in model plants and in the cereal crop barley. The ability to deliver CRISPR plasmids in a non-integrating manner or to achieve DNA-free genome editing through Cas9-gRNA complex delivery would enable faster and more precise mapping of plant traits by avoiding transgene integration and the need for transgene segregation. Furthermore, the abiotic nature of nanoparticle-based DNA or protein delivery may enable facile genetic manipulation and functional genomics in other crops of relevance beyond those tested in the current work. In addition to the above efforts, this proposal will support training of undergraduate students from the Society for the Advancement of Chicanos and Native Americans in Science, and the development of a workshop to teach nanomaterials science to students from a broad variety of backgrounds.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.