A CRISPR/Cas9‐based genome‐editing system for yam (Dioscorea spp.)

[...]we selected promoters DaU6.3 and DaU6.5 to direct the gRNA expression for stable transformation of yam. Black bars indicate exons of the gene, PAM sequences in blue and underlined, and protospacer sequences in red. (d) Schematic presentation of pCas9_gRNA‐PDS used to generate genome‐edited events. (e) Transient gene expression in yam leaves agroinfiltrated with Agrobacterium harbouring pCas9_gRNA‐PDS. (e‐1) Leaf infiltrated with infection medium only, (e‐2&e‐3) leaf infiltrated with Agrobacterium showing bleached patches, (e‐4) microscopic examination of an infiltrated leaf section, (e‐5) green fluorescent micrograph of the infiltrated section, (e‐6) green fluorescent micrograph of an infiltrated section heat treated at 2 dpi and photographed at 4 dpi. The optimized agroinfiltration system with Agrobacterium strain EHA105 harbouring pCas9‐gRNA‐PDS (OD600 = 0.75) suspended in infiltration buffer (Murashige and Skoog medium salts and vitamins, 20 g/L sucrose, 1 mg/L 6‐benzylaminopurine, 0.2 µm CuSO4, pH 5.7) supplemented with 400 μm acetosyringone, infiltrated in the fully expanded young leaves and heat shock treatment at 37 °C for 30 min at 2 dpi showed the highest level of transient gene expression as bleached patches and a bright GFP fluorescence at 4 dpi (Figure 1e). The target region (300‐bp) of DrPDS from individual plants, with 4 leaves per plant separately sampled for DNA, was amplified by PCR using gene‐specific primers and the amplicons were subjected directly to Sanger sequencing.