Physical maps and structures of CRISPR/Cas9 binary vectors. (A) Physical maps of the backbones of pGreen and pCAMBIA from which CRISPR/Cas9 binary vectors were derived. The map of the helper plasmid required for propagation of pGreen in Agrobacterium and the mutated BsaI site on the pCAMBIA backbone are indicated. LB/RB, left/right border of T-DNA; pSa-ori, required for replication in Agrobacterium engineered with the corresponding replication protein (pSa-repA); KmR, kanamycin resistance gene; pUC-ori, replication origin required for replication in E. coli; pVS1-staA, pVS1-ori and pVS1-rep are the DNA elements required for replication in Agrobacterium. Only the 225-bp fragment between the LB and RB was left for comparison of the sizes of the pGreen and pCAMBIA backbones. (B, C) Physical maps of the regions between the RB and LB. The sizes of T-DNA regions and the structures of SpR-gRNA-Sc and final working gRNA are indicated. zCas9, Zea mays codon-optimized Cas9; U6-26p, Arabidopsis U6 gene promoter; U6-26t, U6-26 terminator with downstream sequence; OsU3p, rice U3 promoter; OsU3t, rice U3 terminator with downstream sequence; SpR, spectinomycin resistance gene; gRNA-Sc, gRNA scaffold.