High-efficiency retron-mediated single-stranded DNA production in plants
Type
ArticleAuthors
Jiang, WenjunSivakrishna Rao, Gundra
Aman, Rashid

Butt, Haroon

Kamel, Radwa
Sedeek, Khalid Elwy Mohamed

Mahfouz, Magdy M.

KAUST Department
Bioengineering ProgramBiological and Environmental Science and Engineering (BESE) Division
Bioscience
Bioscience Program
Center for Desert Agriculture
Laboratory for Genome Engineering
Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology (KAUST) , Thuwal 23955-6900, Saudi Arabia
Plant Science
Date
2022-11-01Permanent link to this record
http://hdl.handle.net/10754/673948
Metadata
Show full item recordAbstract
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/ysac025Sponsors
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 Biologyae974a485f413a2113503eed53cd6c53
10.1093/synbio/ysac025
Scopus Count
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