Fig. 4.

Mutations induced by Cas9 paired nickases. (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) Total mutation variations on A2t and/or AB3b sites using Cas9 paired nickases. 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 indicate deleted bases. The net changes in length are shown to the right of each sequence (–, deletion). The number of clones representing each mutant allele is shown in brackets. (c) CAPS analysis of the off-target gene, DMC1B.