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

CRISPR/Cas的出现使得精确编辑基因的能力达到了前所未有的规模。目前实现基因编辑的全部潜力的主要瓶颈是缺乏将基因编辑成分输送到植物中的可靠方法。将基因编辑成分传递到植物细胞中最广泛使用的方法是通过转化。然而，许多主要作物是难以转化的，转基因植物的生产是耗时和劳动密集型的。由于玉米的转化仍然局限于专门的实验室，这是CRISPR/Cas应用于功能基因组研究的主要障碍。在这里，我们将生成资源和开发工具，以简化基因编辑技术在玉米中的应用。鉴于玉米是世界上产量最高的作物，每英亩产生的热量几乎比任何其他作物都要高，该项目开发的工具将对涉及玉米和其他重要单株作物的研究产生更广泛的影响。在这个项目下产生的所有生物材料和方法将提供给科学界。该项目还将为未被充分代表的少数民族本科生和高中生提供实践研究培训。开发更新、更有效的植物基因编辑方法对于进行基因功能研究至关重要。基于CRISPR/ cas技术的出现和部署使得具有高度特异性突变的突变基因型得以产生。到目前为止，大多数高效的基于crispr的基因编辑方法都依赖于传统的转基因方法来传递Cas核酸酶和单导RNA （sgRNA）成分。虽然很少有玉米自交系可以转化，但转化是玉米功能基因组研究的主要瓶颈。此外，传统的自交系有较长的世代时间，需要大量的生长空间。为了克服这些瓶颈，本项目将产生在组成型泛素启动子控制下表达高水平Cas9核酸酶的B104玉米品系。将sgrna传递到这些表达Cas9的玉米系的病毒载体将被设计和优化，以诱导体细胞和遗传编辑。为了加速玉米基因功能的研究，在组成型泛素和减数分裂特异性启动子的控制下，将产生高水平表达Cas9的快速开花迷你玉米（FFMM）系。这些细胞系和用于传递sgrna的优化病毒载体将进行诱导体细胞和遗传编辑的测试。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。

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.