Development of Barley with Heritable Disrupted ENGase Gene via CRISPR-Cas9

Targeted genome editing with the CRISPR-Cas9 system has been used extensively for the selective mutation of plant genes. Researchers from the University of Natural Resources and Life Sciences in Austria, led by Eszter Kapusi, used CRISPR/Cas9 to disrupt the barley (Hordeum vulgare) endo-N-acetyl-β-D-glucosaminidase (ENGase) gene.

Five single guide RNAs (sgRNAs) were designed to target different sites in the upstream part of the ENGase coding region. Genotype screening was carried out in the primary transformants and their progeny to confirm the presence of site-specific small deletions and insertions (indels) and genomic fragment deletions between pairs of targets.

Mutations were observed in 78% of the plants, a higher efficiency than previously reported in barley. The induced indels and fragment deletions were transmitted to the T1 generation, and transgene free genome-edited homozygous ENGase knock outs were identified among the T1 progeny.

This study demonstrated that mutant barley lines with a disrupted ENGase can be produced efficiently using the CRISPR-Cas9 system.

For more on this study, read the article in Frontiers in Plant Science.