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Fig. 3 | Journal of Genetic Engineering and Biotechnology

Fig. 3

From: Multiplex CRISPR/Cas9 gene-editing platform in oil palm targeting mutations in EgFAD2 and EgPAT genes

Fig. 3

Schematic map for assembling two and four sgRNA expression cassettes into a pYLCRISPR/Cas9 vector (a) pYLCRISPR/Cas9P35S-H: EgFAD2 (16,846 bp), (b) pYLCRISPR/Cas9PUbi-H: EgPAT (18,036 bp), and (c) pYLCRISPR/Cas9PUbi-H: EgFAD2EgPAT (19,147 bp). T35S, cauliflower mosaic virus 35S terminator; Hyg, hygromycin phosphotransferase gene; LB, left border of T-DNA; 2xP35S, the 35S promoter of the double cauliflower mosaic virus; Tnos, nopaline synthase gene terminator; PUbi, maize ubiquitin Ubi1 promoter; Cas9, CRISPR-associated protein 9; T-right DNA’s border, abbreviated as RB. Rice (Oryza sativa) U6a promoter; Rice (Oryza sativa) U6b promoter; The GA-L and GA-R primers are used for Gibson assembly of sgRNA expression cassettes, while the flanking primers SP-L, SP-R, PB-L, and PB-R are employed to amplify the linked sgRNA expression cassettes. (d) AscI digestion to ensure that the sgRNA expression cassettes are successfully inserted. Lanes 1a and 1b: pYLCRISPR/Cas9P35S-H: EgFAD2; Lanes 2a and 2b: pYLCRISPR/Cas9PUbi-H: EgPAT; Lanes 3a and 3b: pYLCRISPR/Cas9PUbi-H: EgFAD2EgPAT

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