High-efficiency retron-mediated single-stranded DNA production in plants.

Area of Science:

• Molecular Biology
• Plant Biotechnology
• Genetics

Background:

• Retrons are retroelements producing multicopy single-stranded DNA (ssDNA), utilized in various organisms for applications like genome engineering.
• ssDNA is crucial for homology-directed repair (HDR), a gene editing mechanism, but its delivery in plants is challenging.
• Retron-mediated ssDNA production in plants offers potential for advanced plant biotechnology.

Purpose of the Study:

• To establish and optimize retron-mediated ssDNA overproduction in *Nicotiana benthamiana*.
• To enhance ssDNA production efficiency through novel retron architectures and protective protein co-expression.
• To demonstrate the utility of retron-coupled ssDNA production for targeted HDR in plants.

Main Methods:

• Engineered retron systems for ssDNA production in *Nicotiana benthamiana*.
• Screened various retron architectures to identify optimal designs for increased ssDNA yield.
• Co-expressed retron systems with the ssDNA-binding protein VirE2 and coupled with CRISPR-Cas systems.

Main Results:

• Successfully demonstrated retron-mediated ssDNA overproduction in *N. benthamiana*.
• Identified a novel retron architecture significantly increasing ssDNA abundance.
• Achieved a 10.7-fold increase in *in vivo* ssDNA production by co-expressing VirE2.
• Showcased CRISPR-retron-coupled ssDNA production and targeted HDR in *N. benthamiana*.

Conclusions:

• Developed an efficient *in vivo* method for ssDNA production in plants using retron systems.
• This approach overcomes limitations of synthetic ssDNA delivery for gene editing applications in plants.
• The retron-based technology holds significant promise for advancing plant biotechnology and genetic engineering.