Rapid and efficient gene modification in rice and Brachypodium using TALENs.

Materials and methods TALEN design and plasmid construction All TALEN target sites were identified using the TAL effector-Nucleotide Targeter 2.0 (TALE-NT) program (https://tale-nt.cac.cornell.edu/) (Doyle et al., 2012). TALENs recognizing the target sites were constructed using Golden Gate method as previously described (Cermak et al., 2011; Zhang et al., 2012). The Gateway compatible entry plasmid, pZHY013, was used as the intermediate vector to create TALEN expression vectors (Zhang et al., 2012). This plasmid contained two heterodimeric FokI nuclease domains separated by a T2A translational skipping sequence. TAL arrays in the plasmids pZHY500 and pZHY501 were released by digestion with XbaI/BamHI -one array (left array) was first cloned into pZHY013 as an XbaI/BamHI fragment; the other array (right array) was then cloned into NheI/BglII sites, which had ends compatible with XbaI and BamHI and subcloned into pZHY013 one-by-one (Zhang et al., 2012). A Gateway LR reaction was performed to clone TALEN coding sequences into the destination vector, pGW3, which was the derivative of pMDC32 (Curtis and Grossniklaus, 2003) by replacing the 35S promoter between HindIII and Acc65I restriction enzyme sites with the maize ubiquitin promoter. Each plasmid also contained a marker gene for hygromycin resistance, which was driven by the 35S promoter of cauliflower mosaic virus.