Fig. 1

CRISPR/Cas9-mediated interference with accumulation of the TYLCV genome. a Genome organization of TYLCV. The six partially overlapping ORFs are represented by black filled arrows, and the IR is represented by an open box. The three CRISPR/Cas9 targets are represented by red arrowheads. The sequences of the three targets (IR, CP, and RCRII) are shown on the right. b Schematic representation of the experimental design. Agrobacterium containing engineered tobacco rattle virus (TRV) with sgRNA specific for the TYLCV genome was infiltrated into Cas9-expressing plants. TYLCV was subsequently infiltrated to Cas9OE plants harboring an established TRV infection. Samples were collected at 10–21 days post-infiltration (dpi) for molecular analysis. c Semi-quantitative PCR of TYLCV genomic DNA. TYLCV infiltration into Cas9OE plants harboring IR-sgRNA accumulated lower levels of TYLCV than plants infiltrated with the TRV empty vector. Actin genomic DNA from N. benthamiana was used for normalization. d Assay for rolling-circle amplification (RCA) of the TYLCV genome in plant extracts. Accumulation of TYLCV genomic DNA in plants harboring IR-sgRNA was lower than that in plants inoculated with TYLCV and the TRV empty vector. e Southern blot analysis of TYLCV genomic DNA accumulation in Cas9OE plants. TYLCV genomic DNA was detected with a DIG-labeled probe against a 560-bp sequence within the IR region. All six individual IR-sgRNA-harboring plants that were infiltrated with TYLCV showed lower accumulation of the TYLCV genome than plants inoculated with the TRV empty vector and TYLCV. Arrowheads in (d, e) indicate the expected size of the TYLCV genome. DIG Digoxigenin, M DNA size marker, NB N. benthamiana, PEBV pea early browning virus promoter, RE restriction enzyme