Generation of bbx30 and bbx31 Mutant Using CRISPR/Cas9 Technique

Gene editing using CRISPR/Cas9 technique was performed as previously described (Wang et al., 2015b). Twenty-three-base-pair target sites (5′-N20NGG-3′) within exons of genomic DNA sequences of BBX30 or BBX31 were manually searched and identified, and then each of these sites was evaluated target specificities on the website of potential off-target finder (http://www.rgenome.net/cas-offinder/). Two independent sgRNA target sites of BBX30 or BBX31 were subcloned into pHEE401E vector. These vectors were transformed into Agrobacterium tumefaciens GV3101 by the freeze-thaw method, respectively, and then introduced into Col plants via the floral-dip method (Clough and Bent, 1998). T1 transgenic plants were selected on MS medium containing 25 mg/L hygromycin. The specific mutations in BBX30 or BBX31 were analyzed using gene-specific primers by PCR amplification and sequencing. The pHEE401 transfer-DNA insertion region including CRISPER/Cas9 in bbx30 or bbx31 single mutant at T1 generation was removed by genetic crossing with Col.