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

IF 4.6 2区医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Molecular Therapy-Methods & Clinical Development Pub Date : 2024-10-18 eCollection Date: 2024-12-12 DOI:10.1016/j.omtm.2024.101356

Shao-Chia Lu, Yi-Yuan Lee, Felix G M Andres, Daniel A Moyer, Michael A Barry

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Abstract

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.

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FastAd：快速生成单个腺病毒或多种腺病毒载体文库的多功能工具包。

腺病毒（ads）是体外和体内应用的有效基因传递载体。然而，目前构建腺病毒的方法耗时且效率低下，限制了腺病毒的快速生产和在生成复杂基因文库中的应用。在这里，我们介绍一种快速易用的技术--FastAd，通过两种高效重组酶的协同作用，将重组 "供体 "DNA 直接插入哺乳动物细胞中的感染性 "接收体 "Ads：Cre 和 Bxb1。随后，由此产生的混合重组 Ad 群体要经过 flippase 重组酶的负选择，以去除错过初始重组的病毒。采用这种方法，重组 Ad 的生产时间可从 2 个月缩短到 10 天或更短。FastAd 可用于在 Ad 基因组中插入复杂的遗传 DNA 文库，通过 T25 烧瓶规模转染 300 万个细胞，生成了具有 300 多万个独特克隆的条形码文库，就证明了这一点。此外，我们还利用 FastAd 构建了一个含有全基因组 CRISPR-Cas9 引导 RNA 文库的 Ad 文库，并证明了它在发现新型病毒-宿主相互作用方面的有效性。总之，FastAd 能够快速生成单个 Ad 载体或复杂的基因文库，不仅促进了 Ad 载体的新型应用，也促进了基础病毒学的研究。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Molecular Therapy-Methods & Clinical Development Biochemistry, Genetics and Molecular Biology-Molecular Biology

CiteScore

9.90

自引率

4.30%

发文量

163

审稿时长

12 weeks

期刊介绍： The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella. Topics of particular interest within the journal''s scope include: Gene vector engineering and production, Methods for targeted genome editing and engineering, Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells, Methods for gene and cell vector delivery, Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine, Analysis of gene and cell vector biodistribution and tracking, Pharmacology/toxicology studies of new and next-generation vectors, Methods for cell isolation, engineering, culture, expansion, and transplantation, Cell processing, storage, and banking for therapeutic application, Preclinical and QC/QA assay development, Translational and clinical scale-up and Good Manufacturing procedures and process development, Clinical protocol development, Computational and bioinformatic methods for analysis, modeling, or visualization of biological data, Negotiating the regulatory approval process and obtaining such approval for clinical trials.