Transcription activator-like effector nucleases enable efficient plant genome engineering

Area of Science:

• Plant Biology
• Genome Engineering
• Molecular Biology

Background:

• Precise plant genome engineering holds significant potential for both fundamental research and practical applications.
• Transcription activator-like effector nucleases (TALENs) are powerful tools for targeted DNA modification.

Purpose of the Study:

• To describe and optimize methods for targeted plant genome modification using TALENs.
• To establish efficient TALEN-based gene editing in tobacco (Nicotiana tabacum) protoplasts.

Main Methods:

• TALENs were designed to target the acetolactate synthase (ALS) gene in tobacco.
• TALEN activity was quantified using a protoplast-based single-strand annealing assay with yellow fluorescent protein reporter.
• High-throughput DNA sequencing was used to assess on-target mutation frequencies.

Main Results:

• Optimized TALENs achieved targeted mutations in the ALS gene in 30% of transformed cells.
• Targeted gene insertion frequencies reached approximately 14%.
• Genome modifications were recovered without selection, with 32% of calli showing TALEN-induced ALS mutations, and 4% exhibiting targeted gene replacement.

Conclusions:

• Optimized TALEN reagents and assays are effective for targeted genome modification in plant protoplasts.
• These methods facilitate efficient gene editing in diverse plant species.
• The developed protocols advance the potential of plant genome engineering for basic and applied research.