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-Î?-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.
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