OSCA: A one-step construction of adenovirus system using the Gibson DNA Assembly technology

ABSTRACT Adenovirus (Ad) is a non-enveloped linear double-stranded DNA virus with >50 serotypes in humans. Ad vectors have been used as gene delivery vehicles to express transgenes, siRNAs for gene silencing, or CRISPR/Cas and designer nucleases for genome editing. Although several methods are used to generate Ad vectors, the Ad-making process remains technically challenging and time-consuming. Moreover, the Ad-making techniques have not been improved for the past two decades. Gibson DNA Assembly (GDA) technology allows for one-step isothermal DNA assembly of multiple overlapping fragments. Here, we developed a one-step construction of adenovirus (OSCA) system using GDA technology. Specifically, we first engineered several adenoviral recipient vectors that contain the ccdB suicide gene flanked with two 20-bp unique sequences, which serve as universal sites for GDA reactions in the Ad genome ΔE1 region. In two proof-of-principle experiments, we demonstrated that the GDA reactions were highly efficient, and that the resulting Ad plasmids could be effectively packaged into adenoviruses. Ad-mediated expression of mouse BMP9 in mesenchymal stem cells was shown to effectively induce osteogenic differentiation both in vitro and in vivo. Collectively, our results demonstrate that the OSCA system drastically streamlines Ad-making process and should facilitate Ad-based applications in basic, translational and clinical research.