Fig. 2.

Simultaneous multiplex target editing by ALS-assisted Target-AID. (A) Schematic illustration of EGFP reporter assay. In switch-mEGFP reporter vector, single stop codon inserted immediately downstream of the initiation codon (Top). The switching module, TAG was altered to TAT by Target-AID to express EGFP (Bottom). (B) Fluorescence microscopic images of the rice callus. Expression of EGFP reporter was detected among double transformed callus lines carrying mEGFP and Target-AID. (C) Sequencing chromatograms showing the nucleotide substitutions by Target-AID in switch-mEGFP and ALS genes of calli carrying switch-mEGFP only (Top) and exhibiting EGFP expression and IMZ tolerance after introduced Target-AID vector (Bottom), respectively. Arrowheads with highlighted character indicate the mutation. (D) Sequence alignment of the mutations at mEGFP and ALS loci. Red letters indicate substituted nucleotides.