High-efficiency retron-mediated single-stranded DNA production in plants. Issue 1 (1st November 2022)

• Record Type: Journal Article
• Authors: Jiang, Wenjun; Sivakrishna Rao, Gundra; Aman, Rashid; Butt, Haroon; Kamel, Radwa; Sedeek, Khalid; Mahfouz, Magdy M
• Is Part Of: Synthetic biology. Volume 7:Issue 1(2022)
• Publication Date: 2022-11-01
• Subjects: retron -- ssDNA -- VirE2 -- HDR -- CRISPR-Cas9; Synthetic biology -- Periodicals; 660.6
• DOI: 10.1093/synbio/ysac025
• Languages: English
• ISSNs: 2397-7000

Abstract

Abstract: Retrons are a class of retroelements that produce multicopy single-stranded DNA (ssDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the overproduction of 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 (HDR) 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 overproduction 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 clustered regularly interspaced short palindromic repeats-retron-coupled ssDNA overproduction and targeted HDR in N. benthamiana . Overall, we present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications. Graphical Abstract