S01 - Session P1 - Evaluation of CRISPR-associated nucleases for efficient gene editing using a transient protoplast-based expression system

Authors: Fabiola Guadalu Ramirez Torres *, Rishikesh Ghogare, Amit Dhingra

Advances in gene editing have facilitated new approaches to improving traits of interest in plants. Specifically, CRISPR-Cas9 and associated endonuclease systems allow for targeted modification of genes related to agriculturally important characteristics. SpCas9, for example, works with a tracrRNA:crRNA sequence to target DNA. The RNA duplex tracrRNA:crRNA includes a guideRNA sequence that leads the nuclease SpCas9 to make double-strand breaks. Also, various other endonucleases similar to SpCas9 have been identified from different bacterial species. Other recently identified endonucleases that, while smaller than Cas9 and without a tracrRNA, have been used successfully to edit genes in plants. In order to efficiently use these tools for gene editing, it is necessary to identify ideal plant tissues, which may include embryos, protoplasts or other explants for direct organogenesis, in which to conduct specific editing experiments. We have identified protoplasts as a favorable choice due to the relatively quick acquisition of results and ease of manipulation and transfection. Our work aims to find the most efficient nuclease for gene editing in protoplasts. We are currently testing the ability of various nucleases to edit GFP expressed in Nicotiana benthamiana 16 C Line protoplasts using Sanger sequencing and high-throughput markers. Knowledge of the efficiency of different nucleases and the plant tissues in which they are most effective, is expected to improve the success of future gene editing experiments.