Optimized R2 retroelement complexes for DNA insertion into plant genomes.

Area of Science:

• Plant molecular biology
• Genome engineering
• Biotechnology

Background:

• Traditional Agrobacterium-mediated DNA insertion into plant genomes leads to random integration.
• Current genetic engineering tools like nucleases and prime editors face limitations such as low efficiency, off-target effects, and restricted payload capacity.

Purpose of the Study:

• To adapt the avian Taeniopygia guttata R2 protein (R2Tg) for efficient and targeted DNA insertion into plant genomes.
• To establish a novel R2Tg ribonucleoprotein platform for inserting large DNA payloads into plant genomes.

Main Methods:

• Engineering R2Tg expression cassettes and RNA payloads with intron-disrupted reporters.
• Optimizing ribosomal DNA homology arms and untranslated regions for R2Tg activity.
• Testing the R2Tg system in various plant systems including Arabidopsis thaliana protoplasts, Nicotiana benthamiana leaves, and Solanum lycopersicum seedlings.

Main Results:

• The R2Tg editor system successfully achieved targeted insertion of full-length DNA payloads ranging from 2.2 kb to 5 kb in different plant tissues.
• In Nicotiana benthamiana leaves, targeted integration occurred at an average of 1 copy per genome, demonstrating a 30-fold increase in efficiency over Cas9 homology-directed repair.
• The system utilizes a multicopy genomic safe-harbor site for efficient integration of large genes.

Conclusions:

• The R2Tg ribonucleoprotein platform offers a powerful new tool for targeted DNA insertion in plants.
• This technology enables the efficient addition of multikilobase genes into plant genomes, overcoming previous limitations in payload size and efficiency.
• The R2Tg system represents a significant advancement in plant genome engineering, with potential applications in crop improvement and genetic research.