EAGER: TRTech-PGR: Leveraging DNA ADP-ribosylation for gene editing in plants

The ability to edit (i.e., change) DNA sequences is enabling major breakthroughs in agriculture and medicine, from boosting crop productivity to curing genetic disorders. With current editing tools at hand, only some types of sequence changes are possible to make in DNA. The research community is working tirelessly on expanding the capabilities of DNA editing tools to make a broader array of desired changes. This project is leveraging the recently developed bacterial editing tool, which avoids the common issues associated with traditional tools, and will implement it in plants. The goal of this study is to improve the bacterial editing tool’s efficiency in two model plant species, tobacco and Arabidopsis. Having a more efficient, new plant genome editor on hand would make it possible to generate desired sequence changes in a wide array of crops. Biotechnological innovations realized through this project can help resolve urgent issues that modern agriculture faces by paving the way to the development of superior crop plants that withstand extreme weather events and thus ensuring reliable food supplies for the world’s growing population. In addition to enabling scientific advances, this project aims to introduce plant sciences and biotechnologies to the public through our group’s successful Plants4kids outreach program for children, interactions with K-12 teachers, community guest lectures, student research training, and the development of online content in plant biology and molecular genetics for beginner-level audiences. CRISPR/Cas-based technologies have streamlined genome editing in bacteria, archaea, and eukaryotes and are being broadly adopted in plant sciences. Genome editing can be initiated by different types of targeted DNA damage: double- or single-stranded DNA breaks by Cas nuclease, base deamination by Cas/guide-RNA-linked base editors, base removal by Cas/gRNA-linked DNA glycosylases, and more recently, by introducing a bulky adduct in the DNA by a Cas-linked ADP ribosylase, DarT. Although in all these scenarios the damage to DNA is recognized by the cell and triggers an array of innate DNA repair pathways, the types of edits that the cell makes are determined by the specific repair machinery recruited to the site of damage. Accordingly, for different types of DNA sequence changes, using certain types of editors makes more sense than any others. This project will implement and optimize in tobacco and Arabidopsis the DarT-based “append editing” tool that attaches an ADP-ribose adduct to the last thymidine in the TYT site within the protospacer targeted by nCas9, ultimately leading to a T to A, C or G substitution. The goal of this effort is to improve the editing efficiency of the new tool in plants to the level comparable to that of optimized base editors and deaminases while minimizing off-targeting. This project also aims to make plant sciences and the concepts behind molecular biotechnologies more accessible to the general public, with a variety of in-person and online community engagement activities for different age groups serving to rebuild public trust in science. 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.