Fig. 4

Validating the multiplex guide RNA expression system for generating genome-edited plants. a Indel frequency (%) at three gRNA binding sites in the NaNEC1c gene in protoplasts harboring the pGG-3 vectors. Large deletions are individually calculated at NaNEC1c-g1, -g2, or -g3- binding site. For instance, the large deletion at the NaNEC1c-g1-binding site is calculated by the sum of large deletion occurred between the target sites of -g1 and -g2 and large deletion occurred between the target sites of -g1 and -g3. Error bars represent SD of three replicates (pools of protoplasts). The colors used in the graph represent the different mutation patterns described in b. b Indel frequency (%) in N. attenuata T₀ plants harboring the pGG-3 vectors. c Indel frequency (%) in N. attenuata transformed calli harboring the pGG-4 is calculated by the sum of small indel frequency and large deletion frequency at each gRNA-binding site. Schematic maps of gRNA12, gRNA14, gRNA1, and gRNA2 expression vector. gRNA12 and gRNA14 bind to the first exon of NaEAH1 and gRNA1 and gRNA2 bind to the first exon of NaNEC1c. Large deletions occurred between the target sites of gRNA12 and 14, and between the target sites of gRNA1 and 2. d Indel frequency (%) in N. attenuata calli harboring the pGG-5 vectors. Agrobacterium was used to transform N. attenuata. Total mutation frequency was calculated by targeted deep sequencing