Figure 1.

Targeted insertion of GFP into N′ gene. (a) Schematic diagram of the eGFP donor. The eGFP, 5′‐phosphorylated (indicated with “P‐”) donor harbouring two phosphorothioate‐linkages (indicated with “*”) at both 5′‐ends, was amplified by using chemically modified primers GFP‐1 and GFP‐2. (b) Schematic diagram of N′ gene and eGFP inserted chimeric N′ gene with the targets (underlined), PAM sequences (orange boxed and emboldened letters), cleavage sites (red arrowhead), and primers (arrows). The N′ gene is illustrated in dark green and GFP donor is in light green. (c) GFP fluorescence signals in eGFP donor and Cas9 plasmid transfected protoplasts, and protoplast transfected with only eGFP donor as control. GFP channel, DIC channel and merged channel are single microscope sections. Bar = 100 μm. (d) PCR verification of eGFP insertions. The N′ specific primer N′F1 and the eGFP‐specific GFP‐R1 primer were used for verification of the targeted insertions. The protoplasts were transfected with the donor eGFP and Cas9 plasmids or with only the donor eGFP as a control. Three experiments were performed. Protoplast DNA samples with targeted insertions resulted in a PCR product of ~546 bp, while samples from protoplast transfected with only donor eGFP did not result in target bands.