Indirect Monitoring of Engineered Nuclease Activity for Target Gene Modification in Petunia

Engineered nucleases are an innovative tool for genome engineering. By targeted cleavage of a specific genomic location in cells, engineered nucleases facilitate efficient and precise modification of genomic information. However, targeted cleavage at the plant genomic site often fails, necessitating a screening procedure. Here, we present a reliable and convenient procedure to assess in vivo activity of engineered nucleases in a plant genomic context. We developed a protoplast-based monitoring system based on the engineered nuclease, RGEN. A custom designed RGEN was delivered into petunia protoplasts using PEG. First various combination of cellulose enzymes were tested for petunia. Also, the optimum PEG and guided RNA concentrations were determined for higher transformation efficiency. Currently the transformation rate has been raised up to ~30% when GFP was introduced. An enzymatic assay and deep-sequencing showed RGEN inducing the target gene modification efficiently. Phenotypic modification will be ultimately determined, but the current indirect monitoring system demonstrates that the current gene modification conditions for RGEN will be successfully applied to other crops or genes for genome editing efficiency.