Fig. 3

Evaluation of the multiplex guide RNA expression system for editing single gene in protoplasts. a Structure of a single gRNA-expressing system or two such system. A linker sequence (4-nt) is added in between the gRNA scaffold and the tRNA for Golden Gate assembly. Two gRNAs for each of six genes in N. attenuata were designed for mutagenesis. The coding sequences of the target genes are presented as black pentagons. Each gRNA is shown as a black arrow. Distance between two gRNA-cleavage sites are presented immediately below the black pentagons. b Indel frequency (%) at 12 gRNA binding sites in N. attenuata protoplasts. Indel mutation patterns are divided into three categories: insertion (yellow), small deletion (blue), and large deletion (red). The large deletions occur by simultaneous DNA cleavages at two adjacent gRNA-binding sites. Asterisks indicate statistically significant differences (two-tailed Student t-test, *P < 0.05, **P < 0.01). c Relative percentage of small indel (gray) and large deletions (red) to total mutations for each gRNA binding site. Total mutation frequency was determined by targeted deep sequencing. Error bars represent SD of three or five replicates (pools of protoplast). d Large deletions occur by expressing two gRNAs in the protoplasts. Wild type (WT) sequences of the NaEAH1 gene are shown with gRNA-binding sequences (underlined) and protospacer adjacent motif (PAM) in red. In the below of WT sequence, the total indel frequency is given followed by the frequency of large deletions is in parentheses. Indels are presented in blue (insertion) and as dashes (deletion). Total Indel % is the sum of the frequency of small indels and large deletions. The DNA sequences of target locus are ranked with the large deletion frequency