Fig. 1

Reconstitution of the CRISPR/Cas13a machinery in plants. a Schematic assembly of the plant codon-optimized Cas13a (pCas13a). pCas13a was custom synthesized as four fragments. The F1 (with attL1 and 3x-HA), F2, F3, and F4 (with nls and attL2) fragments were assembled in the cloning vector using a restriction-ligation system. The respective restriction enzymes sites are represented on the top. By LR reaction, pCas13a was moved into the Gateway-compatible binary vector pK2GW7 to make pK2GW7-pCas13a. HA human influenza hemagglutinin epitope tag, nls nuclear localization signal, attL1 and attL2, Gateway cloning recombination sites. b Confirmation of pCas13a expression in transgenic lines. Total protein was extracted from three independent lines of N. benthamiana transformed with pK2GW7-pCas13a. Anti-HA antibody was used to detect HA-tagged pCas13a. The arrow indicates the presence of pCas13a. Wild type (WT) was used as a negative control. c The TuMV-GFP genome with selected targets. The arrowheads indicate the four selected sites. The lower panel represents the target RNA sequences paired with their respective crRNAs. PFS protospacer flanking site. d Expression of the crRNA from the TRV system. The repeat-guide DNA sequences were cloned under the PEBV promoter in RNA2 of TRV for constitutive and systemic expression. e Diagrammatic representation of the pCas13–crRNA–target complex. The targeting complex pCas13a–crRNA (repeat sequence-n28, guide RNA-n28) bound to the RNA target n28 for interference