S01 - Session P1 - Genome editing of the EIN2 gene in Cattleya using the CRISPR/Cas9 system

Authors: Yu-Ying Liu *, Pung-Ling Huang, Yi-Yin Do

Flowers of Cattleya are sensitive to ethylene, resulting in petal senescence. Plants impaired in ETHYLENE INSENSITIVE 2 (EIN2), an ethylene signal transduction protein, are almost insensitive to all aspects of ethylene responses. This research utilized the CRISPR/Cas9 system for genome editing of the EIN2 gene to delay flower senescence in Cattleya Rhyncattleanthe Duh's Smile 'The King' (Rd). Exon 1 to exon 4 of RdEIN2 were cloned by polymerase chain reaction (PCR) with degenerate primers designed according to the amino acid sequences in highly conserved regions among orchids EIN2. The RdEIN2 sequence was analyzed to choose a guide sequence for single guide RNA (sgRNA) used in CRISPR/Cas9. The result of in vitro digestion showed that the ribonucleoprotein complex of sgRNA Rdsg1 and the Cas9 protein cleaved target DNA effectively. The constructed plasmid containing Rdsg1, Cas9 and selection marker gene cassettes was transformed into Cattleya leaf cells by agro-infiltration for transient expression. Genomic DNA of transformed leaves were extracted to perform PCR amplification, high-resolution melting (HRM) curve analysis, quantitative real-time reverse transcription PCR (qRT-PCR), and Sanger sequencing for detection of gene-edited mutants by CRISPR/Cas9. We expect that stable transformation of Cattleya EIN2 mutants edited by CRISPR/Cas9 would be insensitive of ethylene and therefore delay the senescence of Cattleya flowers.