Fig. 8. AS efficiency of the endogenous  APXII  gene in  NtAPSI1 -disrupted tobacco plants. Semiquantitative RT-PCR analysis detected AS variants (sAPX-II, -III, and tAPX-I mRNAs) produced from the endogenous  APXII  gene in genome-edited tobacco lines #13-1 and #17-3, and WT tobacco plants (SR1). Semiquantitative RT-PCR analysis (24–28 cycles) was carried out using sets of primers ( Supplementary Table 1 ) in extracts from the leaves of 4-week-old plants (T2 generation). The positions of primers, except for T4, are similar to those of the spinach  APXII  gene in  Fig. 1a . The T4 primer was designed based on the middle region of exon 5. Amplified products were electrophoresed on a 2% (w/v) agarose gel. Equal loading of each amplified cDNA was determined using the control eukaryotic translation initiation factor 1A (eIF1a) PCR product. M, molecular marker. The experiments were repeated at least 3 times using plants grown independently, and the representative results are shown.