4.2. CRISPR Sal1 Transformation Construct

The CRISPR Sal1 transformation construct was constructed using standard restriction enzyme cloning techniques. The map and sequence of the plasmid is shown in Figure S4. The construct carries three Sal1-targeting guide RNAs (gRNA 5′, gRNA middle, and gRNA 3′) flanked by transfer RNA (tRNA) spacers, which are expressed using the switchgrass Ubiquitin 1 promoter and the nopaline synthase transcription terminator. The Cas9 gene is constitutively expressed in wheat using the maize Ubiquitin 1 promoter and nos transcriptional terminator.