Fig 1.
CRISPR/Cas9-induced mutations at a transgene bar in soybean hairy roots.
(A) Schematic illustrating the targeted bar sequence (blue) and corresponding PAM (red). PCR amplification spanning the target loci was conducted using the primers bar-F and bar-R. Restriction enzyme ApaI 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 ApaI. 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.
Fig 2.
CRISPR/Cas9-induced mutations in the GmFEI2-SP2 target site.
(A) Schematic illustrating the GmFEI2-SP2 target sequence (blue) and corresponding PAM (red). PCR amplification spanning the target loci was conducted using the primers GmFEI2-SP2-F and GmFEI2-SP2-R. The restriction enzyme BslI at the Cas9 cleave 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 BslI. 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.
Fig 3.
Simultaneous edition of two target sites with only one customized sgRNA.
(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 GmFEI2-F/GmFEI2-R and GmFEI1-F/GmFEI1-R, respectively. Restriction enzyme BslI 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 BslI. 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.
Table 1.
Summary of CRISPR/Cas9 genome editing assays in the present study.