Malina Abba, Katigbak Alexandra, Cencic Regina, Maïga Rayelle Itoua, Robert Francis, Miura Hisashi, Pelletier Jerry
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Department of Biochemistry, McGill University, Montreal, Quebec, Canada; Department of Oncology, McGill University, Montreal, Quebec, Canada; The Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, Quebec, Canada.
Methods Enzymol. 2014;546:193-213. doi: 10.1016/B978-0-12-801185-0.00010-6.
The use of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) for targeted genome editing has been widely adopted and is considered a "game changing" technology. The ease and rapidity by which this approach can be used to modify endogenous loci in a wide spectrum of cell types and organisms makes it a powerful tool for customizable genetic modifications as well as for large-scale functional genomics. The development of retrovirus-based expression platforms to simultaneously deliver the Cas9 nuclease and single guide (sg) RNAs provides unique opportunities by which to ensure stable and reproducible expression of the editing tools and a broad cell targeting spectrum, while remaining compatible with in vivo genetic screens. Here, we describe methods and highlight considerations for designing and generating sgRNA libraries in all-in-one retroviral vectors for such applications.
CRISPR/Cas9（成簇规律间隔短回文重复序列/CRISPR相关蛋白）用于靶向基因组编辑已被广泛采用，并被视为一项“改变游戏规则”的技术。这种方法能够轻松、快速地用于修饰多种细胞类型和生物体中的内源性基因座，使其成为用于可定制基因修饰以及大规模功能基因组学的强大工具。基于逆转录病毒的表达平台的发展，可同时递送Cas9核酸酶和单向导（sg）RNA，这提供了独特的机会，既能确保编辑工具的稳定和可重复表达，又能实现广泛的细胞靶向谱，同时还能与体内遗传筛选兼容。在此，我们描述了用于此类应用的在一体化逆转录病毒载体中设计和生成sgRNA文库的方法，并强调了相关注意事项。
