Fig. 2

Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey, introns in white) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold. b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show BsmBI and SpeI resistant PCR fragments amplified from N. benthamiana genomic DNA. d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c). XT1 target site appears in blue and XT2 target site in green. Red letters and dashes indicate insertions and deletions respectively