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.