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High-efficiency retron-mediated single-stranded DNA production in plants
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  • Wenjun Jiang,
  • Gundra Sivakrishna Rao,
  • Rashid Aman,
  • Haroon Butt,
  • Radwa Kamel,
  • Khalid Sedeek,
  • Magdy M. Mahfouz
Wenjun Jiang
KAUST

Corresponding Author:[email protected]

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Gundra Sivakrishna Rao
King Abdullah University of Science and Technology
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Rashid Aman
KAUST
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Haroon Butt
KAUST
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Radwa Kamel
KAUST
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Khalid Sedeek
KAUST
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Magdy M. Mahfouz
KAUST
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Abstract

ABSTRACT Background: Retrons are a class of retroelements that produce multicopy single-stranded DNA (msDNA) 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. However, no studies have shown retron-mediated ssDNA production in plants, which could unlock potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Main methods and major results: 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 HDR in N. benthamiana. Conclusion: We present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications.
26 Nov 2022Published in Synthetic Biology volume 7 issue 1. 10.1093/synbio/ysac025