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. 2015 Oct 9;5:14926. doi: 10.1038/srep14926

Figure 2. VIGE in N. benthamiana.

Figure 2

(A) Schematic diagram of T-DNA region in VIGE vectors pCVA-gRNA and pCVB. Arabidopsis U6-26 gene promoter (U6 promoter) in pCVA-gRNA is used to direct the expression of gRNA for VIGE in transgenic Cas9-expressing plants. NbPDS-targeting guide sequence was shown in red. CR, common region. (B) Agroinfection of transgenic Cas9-expressing N. benthamiana (KQ334) plants with pCVB and pCVA-gRNA::NbPDS (left), but not pCVB and pCVA-scaffold (right), caused photobleached phenotype. Pictures were taken at 12 weeks post agroinfiltration (wpi). (C) RT-PCR to show that gRNA is expressed in pCVA-gRNA:NbPDS/pCVB co-infiltration KQ334 plants but not in pCVA-scaffold/pCVB co-infiltration KQ334 plants. (D) VIGE showed high editing efficiency in pCVA-gRNA::NbPDS/pCVB co-infiltrated KQ334 plants. The NbPDS locus was PCR amplified using genomic DNA from photobleached leaf area of pCVA-gRNA::NbPDS/pCVB co-infiltrated KQ334 plant as well as from control plant. PCR product was then digested with MlyI and subsequently run on a 2% gel. The mutation rate was calculated by dividing the intensity of the uncut band by the intensity of all bands in the lane. (E) DNA sequence of wild type (wt) and mutant versions of NbPDS caused by cleavage by Cas9/gRNA complex and DNA repair. PAM was shown in red, MlyI site was shown in dark red overline and gRNA target site was shown in blue. x2 means two same clones.