The study introduces a novel strategy to enhance the multiplex editing capability of the CRISPR/Cas9 system by leveraging the endogenous tRNA-processing system. The researchers developed a polycistronic tRNA-gRNA (PTG) gene that can produce multiple gRNAs from a single transcript. This approach allows for precise and efficient targeting of multiple genomic sites, significantly improving the efficiency of genome editing in plants. The PTG system was tested in rice protoplasts and stable transgenic rice plants, demonstrating high efficiency in chromosomal fragment deletion and targeted mutagenesis. The method's robustness and broad applicability make it a promising tool for enhancing the capabilities of CRISPR/Cas9 in various organisms.The study introduces a novel strategy to enhance the multiplex editing capability of the CRISPR/Cas9 system by leveraging the endogenous tRNA-processing system. The researchers developed a polycistronic tRNA-gRNA (PTG) gene that can produce multiple gRNAs from a single transcript. This approach allows for precise and efficient targeting of multiple genomic sites, significantly improving the efficiency of genome editing in plants. The PTG system was tested in rice protoplasts and stable transgenic rice plants, demonstrating high efficiency in chromosomal fragment deletion and targeted mutagenesis. The method's robustness and broad applicability make it a promising tool for enhancing the capabilities of CRISPR/Cas9 in various organisms.