Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone. We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines. Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).

中文翻译：

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定制CRISPR Cas9供体载体构建的协议，使用pcDNA3骨架截断哺乳动物细胞中的基因。

在原核生物中发现了成簇的规则间隔的短回文重复序列（CRISPR）RNA引导的自适应免疫系统，可以保护细胞免受外源DNA的侵害。CRISPR Cas9系统已经过修改，并被广泛用作各种生物的基因组编辑工具。在这里，我们提供了使用CRISPR Cas9编辑来截断哺乳动物细胞中基因的详细协议。我们描述了使用Gibson大会与常用pcDNA3载体为骨架的定制供体载体构建。我们描述了一种分步方法来截断使用定制的供体载体的哺乳动物细胞系中的目标基因。我们的方法采用2个引导RNA，即突变的Cas9D10A切口酶（Cas9 = CRISPR相关序列9），以及用于同源重组的定制供体载体，以用可选的新霉素抗性盒（NPTII：新霉素磷酸转移酶II）精确截短目的基因。我们提供了有关如何使用Gibson装配设计和构建定制供体载体（以及常用的pcDNA3载体作为骨架）的详细协议，使研究人员可以获得感兴趣的特定基因修饰（基因截短，基因缺失，表位标记或敲除）。在突变中）。使用G418（遗传霉素）在哺乳动物细胞系中选择突变体，结合多种筛选方法：蛋白质印迹分析，聚合酶链反应和Sanger测序可简化突变体的分离。在几种哺乳动物细胞系中进行了原理验证实验。在这里，我们描述了一个详细的协议，该协议使用CRISPR Cas9基因组编辑以使用常用的表达载体pcDNA3作为供体载体的骨架来截断目标基因。提供用于定制供体载体设计和构建的详细协议将使研究人员能够开发独特的基因组编辑工具。迄今为止，用于CRISPR Cas9自定义供体载体构建的详细协议是有限的（Lee等人在Sci Rep 5：8572，2015; Ma等人在Sci Rep 4：4489，2014）。定制供体载体可商购，但价格昂贵。我们的目标是共享该协议，以协助研究人员进行需要特定应用（特定基因截短，敲入突变和表位标记应用）的定制供体载体的基因研究。