PlantSynBio/TR-Tech-PGR: Targeted Integration of User-Defined DNA in Plants

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The placement of new DNA into plant genomes is both inefficient and imprecise. This inefficiency hampers all approaches to develop new and enhance existing agricultural traits in crops. New technology such as CRISPR/Cas9 nucleases act like molecular “scissors” to cut DNA at specific locations in the genome, improving the precision of plant genome engineering. However, after cutting the DNA, the precise addition of new DNA has remained a grand challenge. This project identifies the missing counterpart to the molecular “scissors”--the molecular “glue” needed to precisely add DNA at sites cleaved by Cas9. Project aims include developing the precise addition of DNA into crop genomes, as well as test how this new genome engineering technology can be used to quickly make a single gene or entire plant stress-responsive. This project will also test key rules governing this system of targeted DNA addition, such as the size and type of the DNA that can be introduced. This enabling technology has the potential to transform the engineering of plant genomes for both research and industry. Lastly, this project will excite middle school students in STEM careers and educate undergraduate students that have not yet had other research experience opportunities.The overarching goal of this award is the production of a usable and accessible toolkit of technology that enables future plant synthetic biology approaches to introduce new abilities into key agricultural plants. First, this project aims to target the integration of new DNA into the genome of the discovery plant Arabidopsis thaliana and crop plants maize and soybean. Second, this project aims to explore the types and limitations of the synthetic cargo DNA that can be delivered to specific regions of the genome. These include the delivery of engineered expression cassettes, inserting epitope tags in-frame with protein-coding genes, and adding new enhancer elements upstream of gene coding regions. Third, this project aims to demonstrate how this system of targeted DNA integration can be used to rewire the transcriptional response and engineer the plant phenotype to overcome environmental stress on either a single gene level or for the entire genome. Lastly, this project aims to adapt the “Anyone Can Be A Scientist” outreach program to the COVID and post-COVID eras and develop a course-based undergraduate research experience based on one of the scientific aims to provide students with their first hands-on authentic biological research experience. All project outcomes including genetic and molecular resources will be made available upon request and through long-term repositories.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.