Stable gene replacement in barley by targeted double-strand break induction

Area of Science:

• Plant science
• Molecular biology
• Genetics

Background:

• Gene targeting is crucial for precision genome engineering in plants.
• Homologous recombination (HR) facilitates gene replacement by integrating transformed DNA to substitute existing sequences.

Purpose of the Study:

• To analyze gene targeting efficiency in barley (Hordeum vulgare).
• To establish a model system for gene replacement using double-strand break (DSB) induction.

Main Methods:

• Utilized a transgenic artificial target locus in barley.
• Induced double-strand breaks (DSBs) using the meganuclease I-SceI.
• Analyzed homology-directed DNA integration and inheritance patterns.

Main Results:

• Achieved successful gene replacement in barley transformants via homology-directed DNA integration.
• Observed stable inheritance of the integrated donor construct as a single Mendelian trait.
• Identified one-sided integration as the mechanism for successful events.

Conclusions:

• Gene replacement in barley can be achieved with frequencies suitable for routine application.
• Codon-optimized nucleases and co-transfer of nuclease/donor constructs enhance gene targeting efficiency.
• This approach, using synthetic nucleases, paves the way for routine precision genome engineering in crops like barley.