Fig. 2

CRISPR/Cas9-mediated targeted cleavage of the TYLCV genome. a Mutation analysis using a restriction site loss assay. The TYLCV IR (560 bp) was analyzed for loss of the SspI recognition site at the targeted locus. The arrow indicates the presence of a 269-bp SspI-resistant DNA fragment only in samples harboring IR-sgRNA, but not in samples harboring the TRV empty vector. b T7EI assay for detecting indels in the RCRII domain of the TYLCV genome. The T7EI assay detected mutations only in RCRII PCR amplicons from plants infiltrated with TRV containing RCRII-sgRNA, but not in plants infiltrated with TRV empty vector. DNA fragments A and B were resolved on a 2 % agarose gel and stained with ethidium bromide. Arrows show the expected DNA fragments. c Alignment of reads from PCR amplicons encompassing the IR region, which were subjected to Sanger sequencing. d Alignment of reads from the PCR amplicons encompassing the RCRII motif, which were subjected to Sanger sequencing. The wild-type (WT) TYLCV sequences are shown at the top. The target sequence is shown in red, the SspI site is indicated by a line, and the protospacer-associated motif (PAM) is indicated in green. This is followed by the various indels, which are indicated by the numbers to the right of the sequence (−x indicates deletion of x nucleotides; +x indicates insertion of x nucleotides; and T > G indicates change of T to G). Arrows indicate the expected sizes of the cleavage products