Targeted gene insertion at the <i>dfr</i> locus.

(A) Schematic representation of HDR-mediated gene insertion at the dfr mutant locus using two sgRNAs: sgRNA-DFR#3 and sgRNA-DFR#4 targeting the dfr deletion junction and exon 6 (E6 yellow box). 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 (P and Ter). Gene targeting (GT) homologous sequences are shown with boxes with a hatched pattern. The NptII gene insertion is represented by a blue box. The primers used for detection and sequencing are shown as dark blue arrows. (B) Regenerating explants on kanamycin selective media after Agrobacterium transformation of the T2 plant DFR64a with a single binary vector carrying two sgRNAs and a DNA repair template containing the DFR sequence and NptII gene. The purple coloured events can be visually identified (red arrow) (C) Purple coloured plantlets regenerating on kanamycin-containing media (D) In vitro regenerated T0 plants with HDR-mediated-gene targeting in the DFR landing pad isolated in a tube. (E) PCR analysis for the detection of the precise HR with the repair template at the 5’ junction, 3’ junction and the presence of the NptII gene with primers GT1F and GT1R in six T0 plantlets which showed anthocyanin pigmentation. WT: wild-type; Neg. control: negative control green plantlet; dfr DFR64a: deleted dfr mutant used for the HDR-mediated experiment; 183, 387, 463, 161, 303 and 524: in vitro samples from T0 plantlets issued from targeted insertion events visually screened for their anthocyanin pigmentation. (F) Long range PCR with primers GT3F and GT3R on two different targeted insertions. WT: wild-type; 463 and 524: samples from T0 plantlets from event number 463 and number 524 (with anthocyanin pigmentation). (G) Sequencing of the 5’ and 3’ junctions at the DFR locus in T0 plantlets with recovered anthocyanin pigmentation for events 161, 183, 303, 387, 463 and 524. The sequence of the 3’ junction was not obtained for event number 183. Coloured lower-case letters indicate the end and the beginning of the homologous sequence used in the DNA donor template. Black upper-case letters show the genomic sequences surrounding the insertion. Mutations in the sequences compared to expected sequence are shown with yellow boxes and bold letters.