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

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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.