Lu Shao-Chia, Lee Yi-Yuan, Andres Felix G M, Moyer Daniel A, Barry Michael A
Division of Infectious Diseases, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.
Department of Computational Biology, Cornell University, Ithaca, NY 14850, USA.
Mol Ther Methods Clin Dev. 2024 Oct 18;32(4):101356. doi: 10.1016/j.omtm.2024.101356. eCollection 2024 Dec 12.
Adenoviruses (Ads) are potent gene delivery vectors for and 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.
腺病毒（Ads）是用于多种应用的高效基因传递载体。然而，目前其构建方法既耗时又低效，限制了它们在生成复杂基因文库中的快速生产和应用。在此，我们介绍FastAd，这是一种快速且易于使用的技术，通过两种高效重组酶Cre和Bxb1的协同作用，将重组“供体”DNA直接插入哺乳动物细胞中的感染性“受体”腺病毒中。随后，通过翻转酶重组酶对产生的混合重组腺病毒群体进行阴性筛选，以去除未发生初始重组的病毒。采用这种方法，重组腺病毒的生产时间从2个月减少到10天或更短。FastAd可用于将复杂的基因DNA文库插入腺病毒基因组，从300万个细胞的T25烧瓶规模转染中产生具有超过300万个独特克隆的条形码文库就证明了这一点。此外，我们利用FastAd构建了一个包含全基因组范围综合CRISPR-Cas9引导RNA文库的腺病毒文库，并证明了其在揭示新型病毒-宿主相互作用方面的有效性。总之，FastAd能够快速生成单个腺病毒载体或复杂的基因文库，不仅促进了腺病毒载体的新应用，也推动了基础病毒学的研究。
