一种利用pcDNA3主干构建自定义CRISPR Cas9供体载体截断哺乳动物细胞基因的方案

IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology
Neftali Vazquez, Lilia Sanchez, Rebecca Marks, Eduardo Martinez, Victor Fanniel, Alma Lopez, Andrea Salinas, Itzel Flores, Jesse Hirschmann, Robert Gilkerson, Erin Schuenzel, Robert Dearth, Reginald Halaby, Wendy Innis-Whitehouse, Megan Keniry
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引用次数: 4

摘要

集群规则间隔短回文重复(CRISPR) rna引导的适应性免疫系统在原核生物中被发现,以保护细胞免受外来DNA的侵害。CRISPR - Cas9系统已经被修改,并在广泛的生物体中用作基因组编辑工具。在这里,我们提供了一种使用CRISPR Cas9编辑截断哺乳动物细胞中基因的详细方案。我们描述了自定义供体载体建设使用吉布森装配常用的pcDNA3载体为骨干。我们描述了一个循序渐进的方法截断感兴趣的基因在哺乳动物细胞系使用定制供体载体。我们的方法采用2种引导rna,突变Cas9D10A缺口酶(Cas9?=?CRISPR相关序列9),以及用于同源重组的定制供体载体,用可选择的新霉素抗性盒(NPTII)精确截断感兴趣的基因。我们提供了一个详细的方案,如何设计和构建一个定制的供体载体使用吉布森组装(和常用的pcDNA3载体作为主干),允许研究人员获得特定的基因修饰感兴趣(基因截断,基因缺失,表位标记或敲入突变)。用G418 (Geneticin)筛选哺乳动物细胞系中的突变体,并结合几种筛选方法:western blot分析、聚合酶链反应和Sanger测序,从而获得流线型突变体分离。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方案,利用CRISPR Cas9基因组编辑截断感兴趣的基因,使用常用的表达载体pcDNA3作为供体载体的主干。为定制供体载体设计和构建提供详细的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,CRISPR Cas9自定义供体载体构建的详细方案有限(Lee等人在Sci Rep:8572, 2015;Ma et al.科学通报,2014(4):44 - 44。定制的供体载体在商业上是可用的,但可能很昂贵。我们的目标是分享这一协议,以帮助研究人员进行遗传研究,这些研究需要定制供体载体用于特定的应用(特定的基因截断,敲入突变和表位标记应用)。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone

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).

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来源期刊
BMC Molecular Biology
BMC Molecular Biology 生物-生化与分子生物学
CiteScore
4.80
自引率
0.00%
发文量
0
审稿时长
>12 weeks
期刊介绍: BMC Molecular Biology is an open access journal publishing original peer-reviewed research articles in all aspects of DNA and RNA in a cellular context, encompassing investigations of chromatin, replication, recombination, mutation, repair, transcription, translation and RNA processing and function.
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