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.