Detection of mutations in the CDKB2 gene in Cas9-, sgRNA-expressing calli. (A) CAPS analysis of the CDKB2 locus. DNA extracted from independent pZK_sgCDKB2-transformed calli was subjected to PCR and subsequent BsiWI restriction enzyme digestion. M, marker; –RE, PCR product without restriction enzyme reaction; WT, BsiWI-digested PCR product of wild-type rice DNA. (B) Representative sequences of mutant alleles identified from Cas9-, sgRNA-expressing calli. The wild-type sequence is shown at the top with the PAM sequence highlighted in cyan and the target sequence in red. Dashes indicate deleted bases. The net change in length is noted to the right of each sequence (+, insertion; – deletion). The number of clones representing each mutant allele is shown in brackets. (C) Schematic representation of the chimeric state of multiply mutated and non-mutated cells in clonally propagated transgenic callus. The high proportion of mutated cells makes it easy to obtain mutated plants. Because mutations occur independently in single cells, various mutants can be obtained from a single Cas9, sgRNA transgenic line.