Fig. 3.

Mutations induced by Cas9 paired nucleases. (a) CAPS analysis of the on-target gene, DMC1A. M, molecular weight marker; –RE, PCR product without restriction enzyme reaction; +RE, restriction enzyme-digested PCR product; WT, non-transformed calli; #1–10, independent transformed calli lines. (b) CAPS analysis of the off-target gene, DMC1B. (c) Mutation variations on A2t and/or AB3b sites using Cas9 nuclease. The wild-type sequence is shown at the top with the PAM sequence in green, and the 20 nt target sequence in red. The blue arrowhead indicates the expected cleavage site. Dashes, deleted bases. The net changes in length are shown to the right of each sequence (+, insertion; –, deletion). The number of clones representing each mutant allele is shown in brackets. (d) Total mutation variations on A1t and/or AB3b sites using Cas9 nuclease.