Collaborative Research: TRTech-PGR: Optimization of Virus-based Delivery of Guide RNAs for Heritable Editing in Maize

合作研究：TRTech-PGR：基于病毒的引导 RNA 递送优化，用于玉米遗传编辑

• 批准号: 2303522
• 负责人: Savithramma Dinesh-Kumar
• 金额: $ 32.2万
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2303522&HistoricalAwards=false
• 关键词: Collaborative; Research; TRTech; PGR; Optimization

The advent of CRISPR/Cas has enabled the ability to precisely edit genes at an unprecedented scale. A current major bottleneck in realizing the full potential of gene editing is lack of robust methods for the delivery of gene editing components into plants. The most widely used method for the delivery of gene editing components into plant cells is through transformation. However, many major crops are recalcitrant to transformation and production of transgenic plants is time consuming and labor-intensive. Since transformation of maize is still limited to specialized laboratories, it is a major impediment in application of CRISPR/Cas for functional genomic studies. Here, we will generate resources and develop tools that will ease application of gene editing technology in maize. Given maize is the world’s most produced crop, yields more calories per acre than almost any other crop, the tools developed under this project will have broader impact on studies involving maize and other important monocot crops. All biological materials and the methods generated under this project will be made available to the scientific community. The project will also provide hands-on research training to underrepresented minority undergraduate students and high school students. Developing newer, more efficient gene editing methods in plants is critical to performing gene function studies. The advent and deployment of CRISPR/Cas-based technology has allowed for the generation of mutant genotypes with highly specific mutations. So far, most methods for highly efficient, CRISPR-based gene editing rely on traditional transgenic approaches to deliver the Cas nucleases and single guide RNA (sgRNA) components. Although few maize inbred lines can be transformed, transformation is a major bottleneck in maize functional genomic studies. Furthermore, traditional inbred lines have longer generation times and require a significant amount of growth space. To overcome these bottlenecks, this project will generate B104 maize lines that express high levels of Cas9 nuclease under the control of a constitutive ubiquitin promoter. Viral vectors for delivery of sgRNAs into these Cas9 expressing maize lines will be engineered and optimized for induction of somatic and heritable editing. To accelerate gene function studies in maize, Fast Flowering Mini Maize (FFMM) lines expressing high levels of Cas9 under the control of the constitutive ubiquitin and meiosis-specific promoters will be generated. These lines and optimized viral vectors to deliver sgRNAs will be tested for induction of somatic and heritable editing.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.

CRISPR/CAS的冒险已经使能够以前所未有的规模精确编辑基因。目前，目前实现基因编辑潜力的主要瓶颈是缺乏将基因编辑成分传递到植物中的可靠方法。用于将基因编辑成分传递到植物细胞中的最广泛使用的方法是通过转化。但是，许多主要农作物是转化的转化和转基因植物的生产是耗时和劳动力密集的。由于玉米的转化仍然仅限于专业实验室，因此它是应用CRISPR/CAS用于功能基因组研究的主要障碍。在这里，我们将生成资源并开发可以减轻玉米基因编辑技术应用的工具。给定玉米是世界上生产最多的农作物，每英亩的卡路里比几乎任何其他作物产生更多的卡路里，因此该项目下开发的工具将对涉及玉米和其他重要的单子病作物的研究产生更大的影响。该项目下生成的所有生物材料及其方法都将提供给科学界。该项目还将为代表性不足的少数族裔本科生和高中生提供动手研究培训。在植物中开发更新，更有效的基因编辑方法对于进行基因功能研究至关重要。基于CRISPR/CAS的技术的冒险和部署允许生成具有高度特异性突变的突变基因型。到目前为止，大多数用于基于CRISPR的基因编辑的方法都取决于传统的转基因方法来传递CAS核和单个指南RNA（SGRNA）组件。尽管很少能转化玉米的近交系，但转化是玉米功能基因组研究中的主要瓶颈。此外，传统的近交系列具有更长的生成时间，需要大量的生长空间。为了克服这些瓶颈，该项目将生成B104玉米线，该玉米线在构成性泛素启动子的控制下表达高水平的Cas9核酸酶。将SGRNA传递到这些表达玉米系列中的病毒矢量将经过设计和优化，以诱导躯体和可遗传的编辑。为了加速玉米中的基因功能研究，将产生在组成型泛素和减数分裂特异性启动子的控制下表达高水平CAS9的快速开花迷你玉米（FFMM）线。这些线条和优化的病毒向量将测试用于诱导躯体和可遗传的编辑。该奖项反映了NSF的法定任务，并使用基金会的智力优点和更广泛的影响评估审查标准，认为通过评估被认为是珍贵的支持。