Figure 4
CRISPR/Cas9-mediated deletion mutation of MtGA2ox10. (A) Gene structure of MtGA2ox10. Target sequences of two guide RNAs, G851 and G907, were designed on exon 3. Relative nucleotide positions of the PAM sites marked in boxes are numbered from the start codon. Restriction sites for BsrD I and Eco105 I are also presented. (B) Restriction maps of the wild type PCR products amplified with the 2347-F and 2905-R primers for genotyping by restriction fragment length polymorphism (RFLP), using BsrD I (left) or Eco105 I (right). (C) T-DNA structure of the Cas9 binary construct G851.907 for the deletion mutation of MtGA2ox10. Two single guide RNAs (sgRNAs) (G907 and G851 under MtU6–8 promoters) were tandem-assembled into the binary pGK3304 vector, which contains the GFP::BAR selection marker and Cas9::NLS under CaMV 35S promoters. (D) PCR-RFLP genotyping of A. rhizogenes-transformed roots harboring G851.907. PCR amplicons from four root samples of the pGK3304 empty vector (NULL) and seven root samples of G851.907 (G851.907) were digested independently by BsrD I (left lane) or Eco105 I (right lane). Note that all the amplicons from the NULL samples were digested to fragments of the expected sizes, as shown in the left margin of the agarose gel. In contrast, none of the amplicons from G851.907 samples were digested, indicating disruption of the restriction sites for BsrD I and Eco105 I. Sample 6 shows an increased amplicon size, presumably due to an insertion. (E) Sanger sequencing chromatograms for the PCR amplicons of the G851.907 KO-1, -2, and -3. The PAM sequence for G851 is denoted in the box and the expected cleavage site (−4 bp from PAM) is marked with an arrow, where mixed peaks appear in the sequencing chromatograms. (F) Sequences of each allele in the G851.907 KO-1, -2 and -3. The PAM sequences for sgRNAs are shown in boxes. Predicted changes in the protein structure by deletion mutations are described on the right.
