Fig. 4: Generation of the tomato RIN mutants with long-range mutations by using CRISPR TiD.
From: Genome editing in plants using CRISPR type I-D nuclease
a The detection of long-range deletion mutations in the SlRIN gene induced by the CRISPR TiD. Gene structure, gRNA positions, and the different primer sets to amplify the mutation were indicated. Numbers show the primer sets (1; 1st PCR, 2; nested PCR). The PCR amplified fragments separated on agarose gels indicate CRISPR TiD induced long-range deletions at the tomato RIN locus in the mutant calli (Micro-Tom, T0 generation). VC; vector control plants, 1–7; the transgenic callus lines. b The PCR amplified fragments separated on agarose gels indicate CRISPR TiD induced long-range deletions at the tomato RIN locus in the mutant shoots (Micro-Tom, T0 generation). WT; wild-type, 1–12; the transgenic shoot lines. The large deletions were detected in the lines #4, 5, 6 and 12. The bands with the same length as those of wild-type were non-specific bands. Arrows indicate the specific bands that were subjected to further sequencing analyses; red arrows indicate the fragment1 and blue arrows indicate the fragment2 as shown in c. Young mutant shoots for tomato RIN (Right; Micro-Tom, T0 generation #6) generated by CRISPR TiD. Bar = 1 cm. c Sanger sequencing using the cloned DNA from the CRISPR TiD transgenic tomato shoots (T0; #4, 5, 6, and 12) indicated the large deletion mutations occurred identically, however, the mutation frequencies were varied in the lines. d The mutation sequences of the cloned DNA from a. The nucleotide positions from the PAM were indicated on the sequence.
