2.10 Vector construction and plant transformation
To generate an OsMYC2 CRISPR/Cas9 mutant, a 20-bp gene-specific sequence pair (OsMYc2-CRISPR/Cas9-F: ggcaACGCGTTGTCGTCCGTCCAA and OsMYc2-CRISPR/Cas9-R: aaacTTGGACGGACGACAACGCGT) was synthesized and annealed to form oligo adaptors. Those adaptors were firstly cloned into the entry vector pOs-sgRNA and then inserted into the gateway destination vector pOs-Cas9 as described (Miao et al. , 2013). The CRISPR/Cas9 plasmids were next introduced into Agrobacterium tumefaciens strain EHA105 and then transformed into plants of NIP background. Positive lines were confirmed by PCR followed with sequencing. Within positive lines, those showing both a frame-shift mutation and an absence of the T-DNA backbone based on PCR using primers for hygromycin B phosphotransferase (Hygro-F: ATGAAAAAGCCTGAACTCACCGCG and Hygro-R: TTGCCCTCGGACGAGTG- CTGG) were identified as homozygous lines. The T3 progeny of homozygous lines were used for analyses. The primers used are listed in Table S1.