The CRISPR-Cas9 system has become a powerful and popular tool for genome editing due to its efficiency and simplicity. Multiplex genome editing is an important feature of the CRISPR-Cas9 system and requires simultaneous expression of multiple guide RNAs (gRNAs). Here we describe a general method to efficiently produce many gRNAs from a single gene transcript based on the endogenous tRNA-processing system. A step-by-step protocol is provided for the design and construction of the polycistronic tRNA-gRNA (PTG) gene. The PTG method has been demonstrated to be highly efficient for multiplex genome editing in various plant, animal, and microbial species.