Protoplast technology enables the identification of efficient multiplex genome editing tools in Phalaenopsis.

Phalaenopsis orchids are popular ornamental plants worldwide. The application and optimization of efficient CRISPR-Cas genome editing toolkits in Phalaenopsis greatly accelerate the development of orchid gene function and breeding research. However, these methods are greatly hindered by the deficiency of a rapid screening system. In this study, we established a fast and convenient Phalaenopsis protoplast technology for the identification of functional genome editing tools. Two multiplex genome editing tools, PTG-Cas9-HPG (PTG, polycistronic tRNA-gRNA) system and RMC-Cpf1-HPG (RMC, ribozyme-based multi-crRNA) system, were developed for Phalaenopsis genome editing and further evaluated by established protoplast technology. We successfully detected various editing events comprising substitution and indel at designed target sites of the PDS gene and MADS gene, showing that both PTG-Cas9-HPG and RMC-Cpf1-HPG multiplex genome editing systems are functional in Phalaenopsis. Additionally, by optimizing the promoter that drives Cpf1 expression, we found that Super promoter can significantly improve the editing efficiency of the RMC-Cpf1-HPG system. Altogether, we successfully developed two efficient multiplex genome editing systems, PTG-Cas9-HPG and RMC-Cpf1-HPG, for Phalaenopsis, and the established protoplast-based screening technology provides a valuable foundation for developing more diverse and efficient genome editing toolkits and facilitating the development of orchid precision breeding.