Fig. 3

pCas13a-processed poly-crRNA mediates interference with TuMV-GFP. a Schematic of the cleavage of poly-crRNA by pCas13a. Rectangles denote the spacer sequences and solid lines denote repeat elements. The predicted cleavage sites are depicted. b Northern blot for pCas13a-mediated cleavage of poly-crRNA. RNA blot from two independent transgenic lines of pCas13a (L-9 and L-6) infiltrated with TRV RNA1 and RNA2 expressing specific poly-crRNA and a ns-poly-crRNA and TuMV-GFP. The RNA blot was probed and detected with a DIG-labeled 28-nt RNA fragment against the repeat sequence. The arrow indicates the processed crRNA (56 nt) in L-9 from both ns-poly-crRNA and poly-crRNA compared to WT N. benthamiana. In L-6, the processed crRNA level from poly-crRNA remained under the detection level. The synthetic crRNA (56 nt) was used as an experimental control. c GFP signal data demonstrating the poly-crRNA-mediated interference against TuMV-GFP. pCas13a transgenic N. benthamiana plants infiltrated with TRV expressing poly-crRNA (three crRNAs) and TuMV-GFP were imaged under UV light for GFP at 7 dpi. The GFP signal of the plants expressing poly-crRNAs were compared to plants expressing ns-poly-crRNA or empty vector control. d GFP quantification of poly-crRNA-mediated viral interference. The GFP signal (n = 5) data were used for the generation of a percentile graph. The GFP signal of the plants inoculated with TuMV-GFP and empty vector control were used as a reference. e Northern blot showing the poly-crRNA-based interference against TuMV-GFP. RNA blots were probed with the DIG-labeled RNA fragment and developed with anti-DIG antibody. The arrow indicates the accumulation of the TuMV RNA genome. f Western blot to confirm the TuMV-GFP interference by poly-crRNA. Protein blots from the poly-crRNA and ns-poly-crRNA were detected for GFP with anti-gfp antibody. ns-poly-crRNA and plants inoculated with only TuMV-GFP were used as control. In (e) and (f), the lower panels were used as loading controls