Fig. 4

PCR-amplified DNA from protoplasts transfected with pYLCRISPRCas9Pubi-H:OsFAD2. A DNA fragments of 502 bp and 200 bp were amplified using FAD2-F and FAD2-R primers. B DNA fragments of 776 bp and 474 bp were amplified from second set PCR using FAD2-F2 and FAD2-R primers. Lane M is Trans2K® Plus II DNA Marker (TransGen Biotech, China), and lanes marked with “+” are positive samples. C PCR products at 474 bp were directly sequenced and analysed using SNAPGENE revealed mutations consisting of deletions in the DNA region between sgRNA1and sgRNA2. The blue background part is the deleted sequences; the first line (sequence of the wild type target gene) and target site in grey boxes were highlighted. D Sequencing chromatogram, the red box is the residue of target 1 (OsFAD2-T1), and the green box is the residue of target 2 (OsFAD2-T2)