[Intellectual Contribution]
[Background]
Fig. 1. Selection of appropriate Cas9 and sgRNA expression construct in rice. (A) Six Cas9 constructs and two sgRNA constructs were transformed into rice calli separately and sequentially. The best combination of Cas9 and sgRNA construct was determined and an all-in-one vector was constructed. (B) Using an all-in-one vector, targeted mutagenesis of drooping leaf (DL) gene was conducted. As a result, bi-allelic mutants with the drooping leaf phenotype were obtained efficiently. |
Fig. 2. Multigene knockout utilizing off-target mutations. sgRNA was designed on the consensus sequence of 4 CDK genes. Mutation frequency in CDKA2 gene, which has 1-nt mismatch at 18-nt from PAM sequence (NGG) was almost the same as that in the on-target gene, CDKB2. When the number of mismatch increases and the position of mismatch comes close to PAM sequence, mutation frequency decreased (CDKA1, CDKB1). |
Fig. 3. Increment of mutation frequency by extension of culture period of Cas9 and sgRNA transformed calli. Mutation in DNAs extracted from one and two months cultured calli with the Cas9 and sgRNA transgenic expression construct was detected by CAPS analysis. In one month cultured calli, a few mutated PCR products were detected whereas in 2 months cultured calli, mutated PCR product was significantly increased. |
[Reference]