This study introduces a novel strategy to enhance the multiplex editing capability of the CRISPR/Cas9 system by utilizing the endogenous tRNA-processing system. The researchers engineered a synthetic polycistronic gene (PTG) that contains tandemly arrayed tRNA-gRNA units. This system allows for the precise and efficient production of multiple gRNAs from a single transcript, which are then used to direct Cas9 to multiple chromosomal targets. The tRNA-processing system, which naturally cleaves both ends of tRNA precursors, was harnessed to excise individual gRNAs from the primary transcript, resulting in the generation of functional gRNAs with desired targeting sequences. The PTG system was tested in rice protoplasts and stable transgenic rice plants, demonstrating high efficiency in multiplex genome editing and chromosomal fragment deletion. In transgenic rice plants, the PTG/Cas9 system achieved up to 100% efficiency in targeted mutagenesis, significantly higher than traditional sgRNA-based approaches. The PTG system not only increases the number of gRNAs and targeting sites but also enhances the mutagenesis efficiency at individual sites, especially when multiple gRNAs are expressed using PTG. The study highlights the potential of the tRNA-processing system as a robust and precise platform for generating multiple gRNAs, which can be applied broadly to improve the targeting capability and editing efficiency of CRISPR/Cas9 toolkits in various organisms. The PTG technology enables simultaneous mutagenesis of multiple genomic loci or deletion of short chromosomal fragments, facilitating more sophisticated Cas9 applications such as targeted mutagenesis, deletion of redundant genes, transcriptional modulation of multiple genes, and site-specific integration. The findings demonstrate that the PTG system significantly improves multiplex editing capability and efficiency, making it a valuable tool for genome engineering in plants and other organisms.This study introduces a novel strategy to enhance the multiplex editing capability of the CRISPR/Cas9 system by utilizing the endogenous tRNA-processing system. The researchers engineered a synthetic polycistronic gene (PTG) that contains tandemly arrayed tRNA-gRNA units. This system allows for the precise and efficient production of multiple gRNAs from a single transcript, which are then used to direct Cas9 to multiple chromosomal targets. The tRNA-processing system, which naturally cleaves both ends of tRNA precursors, was harnessed to excise individual gRNAs from the primary transcript, resulting in the generation of functional gRNAs with desired targeting sequences. The PTG system was tested in rice protoplasts and stable transgenic rice plants, demonstrating high efficiency in multiplex genome editing and chromosomal fragment deletion. In transgenic rice plants, the PTG/Cas9 system achieved up to 100% efficiency in targeted mutagenesis, significantly higher than traditional sgRNA-based approaches. The PTG system not only increases the number of gRNAs and targeting sites but also enhances the mutagenesis efficiency at individual sites, especially when multiple gRNAs are expressed using PTG. The study highlights the potential of the tRNA-processing system as a robust and precise platform for generating multiple gRNAs, which can be applied broadly to improve the targeting capability and editing efficiency of CRISPR/Cas9 toolkits in various organisms. The PTG technology enables simultaneous mutagenesis of multiple genomic loci or deletion of short chromosomal fragments, facilitating more sophisticated Cas9 applications such as targeted mutagenesis, deletion of redundant genes, transcriptional modulation of multiple genes, and site-specific integration. The findings demonstrate that the PTG system significantly improves multiplex editing capability and efficiency, making it a valuable tool for genome engineering in plants and other organisms.