CRISPR/Cas9-induced mutations at a transgene <i>bar</i> in soybean hairy roots.

<p>(A) Schematic illustrating the targeted <i>bar</i> sequence (blue) and corresponding PAM (red). PCR amplification spanning the target loci was conducted using the primers <i>bar</i>-F and <i>bar</i>-R. Restriction enzyme <i>Apa</i>I at the Cas9 cleavage site is underlined. (B) PCR/RE assay to detect CRISPR/Cas9-induced mutations in target loci. Lanes 1–30, PCR products of samples treated with sgRNA: Cas9 were digested with <i>Apa</i>I. Lanes WT and WT*, undigested and digested wild-type controls, respectively. The red arrowhead indicates the undigested bands. The numbers at the bottom of the gels indicate mutation frequencies measured according to band intensities. M, DL2000 ladder DNA marker. (C) Cloning and sequencing of the undigested bands. Deletions and insertions are indicated as dashes and red lowercase letters, respectively. Base substitutions are indicated with green capital letters. The types of indels are indicated in the right column.