Gene targeting in plants: 25 years later

Area of Science:

• Plant Molecular Biology
• Genome Engineering
• Biotechnology

Background:

• Gene targeting (GT) in plants was first achieved in tobacco but remained inefficient due to low homologous recombination (HR) frequencies compared to random integration.
• Overcoming low GT efficiency required advancements in understanding HR mechanisms and developing novel genetic engineering tools over 25 years.

Purpose of the Study:

• To review the historical development and recent advancements in plant gene targeting techniques.
• To highlight the impact of site-specific nucleases on enhancing GT efficiency in plants.
• To discuss the future prospects of targeted genome modifications in crop improvement.

Main Methods:

• Early GT attempts relied on homologous recombination (HR), with some improvements via overexpression of HR proteins or negative selectable markers.
• A breakthrough involved using sequence-specific endonucleases to induce double-strand breaks (DSBs) at target loci, dramatically enhancing GT frequencies.
• Subsequent development of zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas system provided versatile tools for targeted DSB induction.

Main Results:

• Inducing DSBs at target sites significantly increased GT frequencies in plants.
• ZFNs enabled targeted gene modification in tobacco, maize, and Arabidopsis.
• Recent advances include TALENs and CRISPR/Cas systems, expanding the toolkit for precise genome editing.

Conclusions:

• Site-specific nucleases have revolutionized plant gene targeting, overcoming previous limitations.
• The development of diverse endonuclease tools paves the way for routine targeted genome modifications.
• These advancements hold significant promise for accelerating crop genetic improvement.