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. 2015 Jul 20;5:12217. doi: 10.1038/srep12217

Figure 1. Schematic diagram of assembling Cas9/sgRNA construct and selecting target sites in the PtoPDS gene.

Figure 1

(A) Schematic illustrating the four sgRNAs (red lines) targeting the PtoPDS coding sequence. Blue boxes indicate exons; orange lines indicate introns; target sites 2 and 3 are titllingly arranged. F1, F2, R1, R2 indicate binding sites of the primers using for PCR amplification. (B) Schematic view of the method for constructing the expression cassettes of sgRNAs. Left, the backbone of sgRNA that any specific targeting sequence can be inserted between the promoter and the unchanged part of guide-RNA using BsaI. Right, the four promters from Arabidopsis, AtU3b, AtU3d, AtU6-1, AtU6-29 were used to drive the four PtoPDS targeted sgRNAs, respectively. (C) Schematic diagram of the assembling of sgRNAs and Cas9 expression cassettes in a single binary vector for plant stable transformation mediated by Agrobacterium. By the design tails after cutting with BsaI, four sgRNA expression cassettes were ligated into the binary vector sequentially.