Crispr/cas9 genome modification platform for hull-less barley development

Overview

This solution applies CRISPR/Cas9 genome editing technology to barley breeding by targeting the NUD gene, which controls whether barley grains retain their hull after threshing. The wild-type hulled trait is dominant, requiring energy- and labor-intensive pearling (dehulling) before grain can be used for food. By knocking out the NUD gene, the platform produces naked barley lines that eliminate this processing step.

The work addresses a food security challenge in Ethiopia, where barley is a primary food crop. Dehulling is traditionally performed by women, so producing hull-less varieties directly reduces post-harvest labor for women smallholder farmers while improving the efficiency and value of the barley value chain. The approach also demonstrates how genomics-enabled breeding combined with customizable endonucleases can accelerate the introduction of desirable traits in locally adapted landraces.

Technical specifications

• Editing platform: CRISPR/Cas9 RNA-guided endonuclease system for site-directed genome modification
• Target gene: NUD (naked caryopsis) gene in barley; knockout produces hull-less phenotype
• Guide RNA validation: Pre-validation by ballistic DNA transfer to leaf epidermis cells and by protoplast transfection
• Transformation method: Binary vector construction followed by Agrobacterium-mediated barley transformation
• Mutation detection: PCR amplification of target and off-target regions using primers Hv_Nud_F and Hv_Nud_R2, confirmed by Sanger sequencing
• Phenotyping protocol: Hand threshing of five spikes per line, four times each, with grain collection after approximately four months of growth to maturity
• Trait pipeline: Barley landrace selection, target motif selection, genotyping, and progeny phenotyping

Technology readiness level

The platform is currently at a pre-commercial research and validation stage. Validation procedures include gRNA activity pre-validation, Agrobacterium-mediated transformation, mutation detection in primary transgenic plants, and genotyping and phenotyping of progeny. Future validation steps cover end-to-end workflow from landrace selection through binary vector construction and progeny characterization. The technology is positioned for further development and field validation prior to deployment, with partnership opportunities available for co-development, sponsored research, and pilot trials.