Simultaneous edition of two target sites with only one customized sgRNA.

<p>(A) Schematic illustrating the target sequence (blue) and corresponding PAM (red). PCR amplifications spanning the target loci were conducted from an identical DNA template using the specific pair primers <i>GmFEI2</i>-F/<i>GmFEI2</i>-R and <i>GmFEI1</i>-F/<i>GmFEI1</i>-R, respectively. Restriction enzyme <i>Bsl</i>I at the Cas9 cleavage site is underlined. (B) PCR/RE assay to detect CRISPR/Cas9-induced mutations in target sites of the two genes. Lanes 1–30, PCR products from the samples treated with sgRNA: Cas9 were digested with <i>Bsl</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. The red frame indicates two simultaneous gene-editing events occurring in the same hairy root. (C) Cloning and sequencing of the undigested bands. Deletions and insertions are indicated as dashes and red lowercase letters, respectively. The types of indels are indicated in the right column. The numbers before brackets correspond to the numbers of the lanes.