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. 2023 Sep 25;40(3):201–209. doi: 10.5511/plantbiotechnology.23.0611a

Figure 2. (A) Schematic representation of the construction of the artificial nuclease gene using the CRISPR/dMac3-Cas9 system. The case of construction of an artificial nuclease gene with three gRNAs for the GBSS1 gene is shown. Chemically synthesized DNAs for the gRNAs were inserted into the BbsI site downstream of the AtU6 promoter. The resultant gRNA genes were located in tandem in pBS_GwIsceI, and they were transferred to pZD-dxCas9 to create a CRISPR/dMac3-Cas9 plasmid, pZD-GBSS-dxCas9_123. (B) Structure of a CRISPR/Cas9 plasmid pZD-GBSS-dxCas9_13, which was constructed by introducing gRNA1 and gRNA3 using multi-gateway method without the integration operation of gRNA2 and otherwise by the similar way.

Figure 2. (A) Schematic representation of the construction of the artificial nuclease gene using the CRISPR/dMac3-Cas9 system. The case of construction of an artificial nuclease gene with three gRNAs for the GBSS1 gene is shown. Chemically synthesized DNAs for the gRNAs were inserted into the BbsI site downstream of the AtU6 promoter. The resultant gRNA genes were located in tandem in pBS_GwIsceI, and they were transferred to pZD-dxCas9 to create a CRISPR/dMac3-Cas9 plasmid, pZD-GBSS-dxCas9_123. (B) Structure of a CRISPR/Cas9 plasmid pZD-GBSS-dxCas9_13, which was constructed by introducing gRNA1 and gRNA3 using multi-gateway method without the integration operation of gRNA2 and otherwise by the similar way.