S01 - Session P1 - Establishment of a CRISPR/Cas9 system for editing of the EIN3 gene in Oncidesa

Authors: Yi-Yin Huang *, Pung-Ling Huang, Yi-Yin Do

The biotechnical approach provides a rich resource for biological research as well as crop breeding by increasing the precision and efficiency for genome editing. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) reduces costs and saves time in the editing of a genome in a specific region and is applicable to various organisms, including plants. Here, we chose guide sequences in the ETHYLENE INSENSITIVE 3 (EIN3) /EIN3-LIKE (EIN3/EIL) gene OgEIL1 for sgRNA and assembled in vitro -transcribed sgRNA with Cas9 endonuclease to form ribonucleoprotein (RNP). The guide sequence-containing DNA fragment amplified from the Oncidesa genome was digested by RNP in vitro . To proof the editing efficiency in vivo , the sgRNA was constructed into an expression plasmid, which consisted of a Cas9 cassette and reporter gene. The CRISPR/Cas9 expression plasmid was transformed into Oncidesa protoplasts by polyethylene glycol for transient assay. Fluorescence emitted by the green fluorescence protein (GFP) reporter gene transformed into protoplasts was observed under a microscope. High resolution melting curve (HRM) analysis and PCR-sequencing showed that a single nucleotide was mutated in the target sites of the Oncidesa genome and the editing efficiency was 1.87-3.3%. Collectively, mutations generated at the OgEIL1 target site by CRISPR/Cas9 has confirmed the applicability of this mutagenesis system in Oncidesa .