The CRISPR system is composed of a Cas9 endonuclease ( Cas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, Cas9 proteins could cleave as many targeted loci as gRNAs bind in a genome. We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a Cas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco ( ) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by Cas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites. This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.