Fig. 4: CRISPR/Cas9-mediated knockout of two BrOG1 homeologs.

a Detection of the target efficiency of sgRNAs in vitro. The DNA fragment indicates the in vitro transcription template of sgRNA (T7 promoter, target, and the sgRNA scaffold sequence). The red and green arrows indicate cut bands and uncut bands, respectively. The “+” and “−” symbols indicate that the corresponding substances were included and omitted, respectively. The negative controls NC-1 and NC-2 indicate the exclusive addition of the DNA fragments of sgRNA5 and g2, respectively. The g1 and g2 positive controls indicate standard gRNA1 and gRNA2, respectively. b Schematic diagram of the T-DNA region of the assembled SaCas9/sgRNA expression vector (VK005-101) for Agrobacterium-mediated transformation. RNA-guided Staphylococcus aureus Cas9 nuclease system (SaCas9) using both the A. thaliana ubiquitin 6-26 (atU6) promoter and a 2× 35S promoter was used to express a single guide RNA (sgRNA) scaffold. For plant selection, the constructs harbored a hygromycin (Hyg) resistance cassette. LB: left border, RB: right border. c Results of agarose gel electrophoresis for the detection of exogenous T-DNA insertion in six T₀ plants. GT-24 indicates the nontransgenic control and P1–P6 indicate different lines in the T₀ generation. Amplified bands of mutants carrying the transgene insertion occurred at 693 bp. d Four mutant alleles that were from one double heterozygous T₀ plant (P2) generated by CRISPR/Cas9 were tested through Sanger sequencing. The name of the allele and the size of each insertion are shown on the right. The inserted base of the alleles is highlighted using the red color. WT indicates a GT-24 nontransgenic plant. The PAMs are underlined. e Sequencing of targeted mutations in transgenic T₀ plants (P1, P4, P5, and P6). The size of each insertion and deletion of the alleles are indicated on the right; WT represents the absence of a mutation. f Detection of exogenous T-DNA insertions in T₁ plants by PCR. Nos. 1–30 represent individual T₁ plants from the inbreeding generations of line P2. g Alignment of the CRISPR/Cas9 target sequences from BrOG1A and BrOG1B compared to the sequence of a potential off-target site. The PAMs are underlined and SNPs are highlighted in red