FastAd: A versatile toolkit for rapid generation of single adenoviruses or diverse adenoviral vector libraries.

Adenoviruses (Ads) are potent gene delivery vectors for in vitro and in vivo applications. However, current methods for their construction are time-consuming and inefficient, limiting their rapid production and utility in generating complex genetic libraries. Here, we introduce FastAd, a rapid and easy-to-use technology for inserting recombinant "donor" DNA directly into infectious "receiver" Ads in mammalian cells by the concerted action of two efficient recombinases: Cre and Bxb1. Subsequently, the resulting mixed recombinant Ad population is subjected to negative selections by flippase recombinase to remove viruses that missed the initial recombination. With this approach, recombinant Ad production time is reduced from 2 months to 10 days or less. FastAd can be applied for inserting complex genetic DNA libraries into Ad genomes, as demonstrated by the generation of barcode libraries with over 3 million unique clones from a T25 flask-scale transfection of 3 million cells. Furthermore, we leveraged FastAd to construct an Ad library containing a comprehensive genome-wide CRISPR-Cas9 guide RNA library and demonstrated its effectiveness in uncovering novel virus-host interactions. In summary, FastAd enables the rapid generation of single Ad vectors or complex genetic libraries, facilitating not only novel applications of Ad vectors but also research in foundamental virology.