Efficient Multiplex Genome Editing Tools of Protoplast Technology in Phalaenopsis

Here we established a seedling-leaf protoplast-based platform to identify functional and efficient multiplex genome editing systems for Phalaenopsis. Our data indicated that: (ⅰ) tRNA-based multiplex genome editing tool was effective in Phalaenopsis; (ⅱ) optimizing the sgRNA structure could further improve the efficiency of the tRNA-based multiplex genome editing tools in Phalaenopsis; (ⅲ) dual Pol II promoter-Cpf1 multiplex genome editing system was also functional in Phalaenopsis. The newly established protoplast platform and the multiplex genome editing tools for Phalaenopsis, have great application potentials in rapidly constructing large-scale mutant libraries of orchid and speeding up orchid precise breeding in the future.