Figure 2.

Polymerase chain reaction (PCR)/T7 endonuclease products for six independently transformed plants (T₁ generation) containing a Cas9 transgene with an sgRNA (single guide RNA) targeting the eIF(iso)4E locus. Transformation with a Cas9 transgene with no sgRNA (Cas9 only) and a non‐transformed wild‐type (WT) plant were used as controls. L denotes a 100‐bp DNA ladder. (A) A 1% agarose gel showing multiplex PCR products confirming the presence/absence of the Cas9 transgene, using the constitutively expressed house‐keeping gene EF1a as a loading control. (B) A 1% agarose gel showing PCR amplicons spanning the putative mutation site at the eIF(iso)4E locus. (C) A 2% agarose gel showing eIF(iso)4E cleavage products after a self‐annealing reaction of the PCR amplicon, and subsequent digestion with T7 endonuclease. The presence of the 378‐bp cleavage product in samples 1, 3 and 6 is indicative of Cas9‐induced mutation at the eIF(iso)4E locus (the corresponding 196‐bp cleavage product is not visible on the gel).