Fig. 3
Strategy for the construction of pYLCRISPR/Cas9 targeting OsFAD2-1 in rice. A Schematic illustration of target sites for two sgRNA expression cassettes driven by U6 promoters from rice; OsU6a promoter for OsFAD2-T1 and OsU6b promoter for OsFAD2-T2, the 20-nt sequences in green square boxes and orange square boxes indicate the PAM motif of each sgRNA, the BsaI restriction enzyme sites for the entry of multiple sgRNA expression cassettes, SP-L2 and SP-R are sequencing vector-specific primers, GA-L and GA-R are GA site-specific primers for the Gibson assembly of sgRNA expression cassettes; B pYLCRISPR/Cas9PubiH basic vector has two BsaI sites for insertion of two sgRNA expression cassettes [17] (LB: left border of T-DNA, T35S: cauliflower mosaic virus 35S terminator, HPT: gene for hygromycin phosphotransferase, 2xP35S: Double cauliflower mosaic virus 35S promoter, Pubi: maize ubiquitin Ubi1 promoter, NLS: Nuclear localization sequence. Cas9p: CRISPR associated protein 9, Nos: nopaline synthase gene terminator, RB: right border of T-DNA, pVS1 replicon: pVS1 for replication, pBR322: pBR322 vector, KanR: Kanamycin resistance gene)