Fig. 2

Schematic of binary plasmid vectors delivered to barley and B. oleracea. Transcription units were assembled into the binary plasmid backbone pAGM4723 or pAGM8031 using Golden Gate Modular Cloning. a The barley constructs, sgRNAHvPM19-1 and sgRNAHvPM19-3 house a hygromycin resistance cassette consisting of the hygromycin phosphotransferase coding sequence (hptII) driven and terminated by the 35 s promoter (P-CaMV35s) and terminator (T-CaMV35s) from Cauliflower mosaic virus; a Cas9 expression cassette consisting of sequence encoding Cas9 from Streptococcus pyogenies with a carboxy-terminal nuclear-localization signal from Simian vacuolating virus 40 (SpCas9:NLS) driven by a ubiquitin promoter from Zea mays (P-ZmUbi) and terminated by a nopaline synthase terminator from Agrobacterium tumefaciens (T-AtNos); and single guide RNA (sgRNAHvPM19-1 or sgRNAHvPM19-3) driven by a Triticum aestivum U6 promoter (P-TaU6). b The Brassica construct, sgRNABolC.GA4.a, houses a kanamycin resistance cassette consisting of the neomycin phosphotransferase coding sequence (nptII) driven and terminated by P-CaMV35S and T-AtNos; SpCas9:NLS driven by a constitutive promoter from Cassava Vein Mosaic Virus (P-CsVMV) and a tandem pair of single guide RNAs (sgRNA1BolC.GA4.a and sgRNA2BolC.GA4.a) driven by the U626 promoter from Arabidopsis (P-AtU626)