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

Abstract
Retrons are a class of retroelements that produce multicopy single-stranded DNA and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the over-production of single-stranded DNA (ssDNA), genome engineering, and directed evolution in bacteria, yeast, and mammalian cells. Retron-mediated ssDNA production in plants could unlock their potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Here, we demonstrated retron-mediated over-production of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated CRISPR-retron-coupled ssDNA over-production and targeted homology-directed repair in N. benthamiana. Overall, we present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications.

Citation
Jiang, W., Sivakrishna Rao, G., Aman, R., Butt, H., Kamel, R., Sedeek, K., & Mahfouz, M. M. (2022). High-efficiency retron-mediated single-stranded DNA production in plants. Synthetic Biology. https://doi.org/10.1093/synbio/ysac025

Acknowledgements
This work is supported by KAUST baseline funding to MM. We would like to thank members of the genome engineering and synthetic biology laboratory at KAUST for their critical discussion and technical help in this work.

Publisher
Oxford University Press (OUP)

Journal
Synthetic Biology

DOI
10.1093/synbio/ysac025

Additional Links
https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac025/6786268

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2022-11-03 05:50:13
Accepted in Journal
2021-12-08 10:29:35
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