DBD Jet Delivery for Rapid CRISPR/Cas9 Editing in Plants

Gene editing in crop plants commonly employs clustered regularly interspaced short palindromic repeats (CRISPR) technology. After insertion of an RNA-guided Cas9 nuclease into plant cells, the CRISPR/Cas9 system precisely cleaves DNA and through endogenous molecular repair mechanisms induces gene insertions or deletions within the plant, allowing for precise gene validation studies. There currently is not an effective delivery mechanism for the CRISPR/Cas9 system that enables \textit{in vivo} plant transformation, as existing methods require a lengthy \textit{in vitro} tissue culture and regeneration process. This project tests a novel CRISPR/Cas9 delivery system, using a non-thermal atmospheric-pressure dielectric barrier discharge (DBD) jet to transfer CRISPR/Cas9 plasmids and ribonucleoproteins into rice tissues for rapid gene editing. This is based on a common microbiology technique called electroporation, wherein a high electric field near the cell induces pores to open in the cellular membrane. Here we test the ability to induce pores in the significantly thicker plant cell wall as well, which will enable \textit{in vivo} plant transformation. This technique shows promise to significantly shorten the transformation time for gene editing in plants.