Two-step cloning system for multiplex guide RNA expression in plants. a Cloning procedures of multiplex guide RNAs (gRNAs). b The single gRNA-cloning vector, pGRNA, is designed for a PCR-free multiplex gRNA cloning method. The target-binding sequence of gRNA is prepared by annealing two complementary oligos. A pair of annealed oligos is directly cloned into the BsaI-digested pGRNA (blue triangles) between the tRNA sequence and gRNA scaffold. Two AarI sites (red triangles) are used in step 2. c The Golden Gate assembly for preparing a plant binary vector expressing five gRNAs under a single U6 promoter. Each tRNA-gRNA unit is excised from pGRNA by cutting the pGRNA with AarI. All tRNA-gRNA units and one of the plant binary vectors—pECO100, pECO200, or pECO300—is connected in the Golden Gate assembly mixture described in "Methods"