Generation of KARRIKIN INSENSITIVE2 loss-of-function mutants in Ceratopteris richardii using a CRISPR/Cas9 system based on ribozyme-gRNA-ribozyme (RGR) technology

Plant hormones regulate almost every aspect of plant growth and development. KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling, which is thought to transduce signals derived from an unidentified ligand known as the KAI2 ligand (KL), regulates numerous traits, including seed germination in angiosperms and vegetative reproduction in bryophytes. The origin of KAI2 is believed to be ancient, and the evolution of its signaling pathways remains of significant interest. Ferns represent critical lineages for elucidating the evolution of land plant traits and growth mechanisms that enabled adaptation to terrestrial environments. Therefore, functional studies of key components of this pathway in ferns are essential for understanding the evolutionary trajectory of KAI2-dependent signaling during vascular plant diversification. However, experimental platforms for the CRISPR/Cas9 system, a powerful tool for investigating gene function, remain undeveloped in ferns. Here, we report an efficient CRISPR/Cas9 system based on ribozyme-gRNA-ribozyme (RGR) technology in the model fern, Ceratopteris richardii ( C. richardii ). We generated loss-of-function mutants of the KAI2 ortholog in C. richardii ( CrKAI2 ), as well as the signaling components CrMAX2 and CrSMXL . We demonstrate that exogenous application of an artificial KL agonist increases the expression of KAI2-dependent signaling responsive genes in wild type plants; this response is abolished in Crkai2 mutants. These findings indicate that KAI2-dependent signaling is conserved in C. richardii . Furthermore, this study proposes an efficient CRISPR/Cas9 method that will facilitate genetic studies in ferns.