Fig. 5
From: A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard
Second dimension Multiplexing with Goldenbraid. a Pipeline of the 2D multiplexing strategy. Targets are designed as level 0 structures and combined with standard level −1 parts to create individual oligomers that are combined in level 1 polycistronic tRNA–gRNA structures. The binary combination of two polycistrons incorporates a 2D multiplexing step on the CRISPR cloning workflow. b Restriction analysis of two clones of level 1 polycistronic structures targeting fucosyl (Lanes 1 and 2; EcoRI expected bands: 6345-796) and xylosyltransferases (Lanes 3 and 4; HindIII expected bands: 6345-623), two clones of a level 2 construct derived from their binary assembly (Lanes 5 and 6; BamHI expected bands: 6674-1401) and two clones of its assembly with the hCas9 (Lanes 7 and 8; BsmBI expected bands: 7215-6367)