The GE-NUD barley line was generated by CRISPR-Cas editing of the ethylene response transcription factor gene nud in Hordeum vulgare.

nud is the single causal gene of the covered phenotype and hull adhesion in barley. The intended phenotype of the GE-NUD barley line was a naked (hull-less) caryopsis.

CRISPR-Cas gene editing was performed with disarmed Agrobacterium tumefaciens harboring the plasmid construct HB0193. The construct HB0193 contained two guide RNAs targeting 20bp from exon 1 or 20bp from exon 2 of the nud gene, the gene cassette encoding CRISPR-Cas9 and the selectable hpt gene.

Partial deletion of the NUD coding sequence includes a single base pair deletion in the NUD coding sequence confirmed via Sanger sequencing.

PCR using primers designed on the hygromycin selectable marker and the Cas9 coding sequence was used to confirm absence of the selectable marker, Cas9, and plasmid backbone in the mutant line.

The GE-NUD barley line shows hull-less caryopses. The edited line will be used to conduct a field trial to assess the effect of naked caryopsis on agronomic, malting quality, and brewing quality traits.

Source:

USDA APHIS letter of inquiry