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

全球可持续性努力依赖于植物育种技术的改进，特别是谷类作物等与人类消费相关的作物育种技术的改进。驯化植物的野生近缘种含有许多能使植物抵抗压力的基因。然而，由于鉴定植物抗逆性基因所需的实验通量低，我们鉴定这些基因并将其功能分配给植物抗逆性的能力一直很困难。限制作物遗传抗逆性测试吞吐量的主要瓶颈是将生物分子传递到植物细胞中的能力，其中植物细胞壁对于引入植物遗传作图所需的分子生物学工具来说是一个很大的不可逾越的屏障。在这个项目中，纳米粒子将被开发用于将遗传物质输送到植物细胞中，重点是谷类作物大麦。这些纳米颗粒将被化学功能化，以将基因组编辑货物运送到植物中，这将使探测与植物抗逆性相关的特定植物基因成为可能。该项目将寻求确定哪些基因使谷类作物能够抵御环境压力，并利用这些知识来了解与全球粮食供应相关的其他作物的耐受性。上述研究还将支持代表性不足的本科生作为纳米材料合成和表征的研究实习生，并将开发一个关于纳米材料科学的学生研讨会。研究植物基因型与表型之间的关系对于鉴定植物生物和非生物胁迫基因至关重要。具体地说，作物野生近缘种含有赋予作物适应气候变化相关性状的基因。通过功能基因组学绘制基因型与表型之间的关系，从CRISPR基因组编辑技术的出现中受益匪浅。然而，植物基因组编辑的效率限制了该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基于crispr的DNA质粒的物理化学吸附，以及Cas9-gRNA核糖核蛋白复合物。这些纳米颗粒-生物分子货物复合物将在模式植物和谷类作物大麦中测试其递送和基因组编辑功效。以非整合方式传递CRISPR质粒或通过Cas9-gRNA复合物传递实现无dna基因组编辑的能力，将避免转基因整合和转基因分离，从而实现更快、更精确的植物性状定位。此外，基于纳米颗粒的DNA或蛋白质传递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，而不是在当前工作中测试的那些。除了上述努力之外，该提案还将支持对奇卡诺人和印第安人科学进步协会的本科生进行培训，并开发一个讲习班，向来自各种背景的学生教授纳米材料科学。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.