Fig 1. CRISPR-Cas9 mediated deletion in DFR marker gene.

(A) Schematic illustration of the double sgRNA strategy targeting the DFR gene in exon 3 and exon 6 (E3 and E6 yellow boxes). The PAM site is in green, the target sequence in red and the sgRNA in blue. The promoter and terminator sequences are represented with pale blue and orange boxes, respectively. The expected length of the deletion generated by the use of these two sgRNAs is 1013 bp. The positions of the primers used for detection and sequencing are shown with dark blue arrows. (B) PCR analysis of the DFR locus using primers DD1F and DD1R in 13 independent T0 plants. The amplicon sizes for the wild-type genomic locus and for the predicted deletion are 2360 bp and 1347 bp, respectively (see arrows). The presence of the CAS9 gene in the T0 plants was confirmed by PCR analysis using primers Cas9F and Cas9R. (C) Schematic representation of the deletion pattern observed in the DFR gene for the 13 T0 plants analysed. The numbers inside the grey section represent the deletion size and the numbers next to the blue sections represent the position of the cut in the DFR gene. (D) Emerging hypocotyls observed on 10 day old progeny of a T0 tomato plant that was heterozygous for the DFR gene deletion. Segregating T1 plantlets homozygous for the mutated DFR gene showed an absence of anthocyanin pigmentation in their hypocotyls (white arrow). Segregating T1 plantlets homozygous or heterozygous for the wild-type DFR gene showed anthocyanin pigmentation (red arrow). (E) Regeneration from cotyledon pieces two weeks after induction. Left: cotyledons of T2 plantlets issued from the dfr mutant T0 plant DFR64a; Right: cotyledons of wild-type WVA106. Anthocyanin pigmentation (red arrow) was observed in the first days of regeneration while it was not visible (white arrow) in dfr regenerating buds. (F) Anthocyanin content analysis (μg equivalent cyanidin-3-glucoside per g of fresh weight) by high-performance liquid chromatography on cotyledons and regenerating plantlets in wild type and dfr mutants. Coty WT: WVA106 wild-type cotyledon extract; Coty mut: dfr mutant extract; Rege WT: wild-type regenerating plantlet extract; Rege mut: dfr mutant regenerating plantlet extract. Anthocyanin contents were not detected in the mutant extracts with a minimal level of detection of 0.05 μg/g.