TRTech-PGR: Development of highly efficient and unconstrained CRISPR systems for plant functional genomics

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Emerging plant genome engineering technologies are tools designed to expand basic research and translational research. In recent years, different genome editing tools have been developed for generating targeted mutations in the plant genome with single base precision. Since agronomic traits are caused by mutations or DNA variation, genome editing technologies accelerate the breeding of high yield, nutritious, disease resistant, and climate resilient crops to meet the demand of an increasing world population and evolving diet. Genome editing technologies are often used to inactivate or alter gene function. By contrast, gene activation technologies can enhance gene function, which provide new means for metabolic engineering and crop improvement. Currently, these genome engineering tools have limited targeting scope, making many genomic or gene sequences inaccessible for editing or activation. To overcome this major bottleneck, we will develop a suite of improved genome engineering tools that provide more flexibility for targeting and are of high efficiency and precision. Furthermore, we will convert these new tools into a comprehensive all-in-one swiss knife-like toolbox so that users can pick appropriate tools for different genome engineering applications in plants. Such a toolbox will not only accommodate small scale usage when targeting only one gene at a time, but also facilitate large-scale genome-wide screens for trait discovery in model plants and crops. Training future scientists, especially from historically under-represented groups, is a core mission of this research project. Plant researchers have greatly benefited from the rapidly evolving CRISPR-Cas9, Cas12a, C-to-T base editing and A-to-G base editing systems. CRISPR-Cas9 has also been repurposed and engineered into efficient gene activation systems which holds great promise in plant genomics, metabolic engineering, and crop improvement. However, there are two major limitations. The first limitation is genome targeting restriction defined by the protospacer adjacent motifs (PAMs) of Cas9 or Cas12a. The second limitation is a lack of versatile CRISPR vector systems tailored for genome-wide genetic screens in plants. To overcome these two major limitations, this project aims to (1) improve PAM-relaxed genome editing by CRISPR-Cas12a in plants, (2) Improve PAM-less genome editing by CRISPR-Cas9 in plants, (3) Improve PAM-less base editing systems in plants, and (4) Convert new CRISPR systems into all-in-one vectors for large-scale screens in plants. These new genome engineering tools will be tested in both monocot (e.g., rice) and dicot (e.g., tomato) plants, which ensure the technologies’ applicable to all plants. These user-friendly CRISPR vectors will be made available to other researchers through Addgene (www.addgene.org), to help advance basic and translational research in plants. The broader impacts of this project include training for undergraduate students at University of Maryland at College Park and Howard University at Washington (a premier HBCU). The project also brings STEM training to students from Montgomery Blair High School and Eleanor Roosevelt High School (ERHS), which enrolls 60-62% African Americans and 9-12% Hispanics.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.