Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double-Stranded DNA Donors.
Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double-Stranded DNA Donors.
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使用双链 DNA 供体通过基因组工程用荧光报告基因快速标记人类蛋白质。
DOI：
10.1002/cpmb.102
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发表时间：
2019
影响因子：
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通讯作者：
Seydoux,Geraldine
中科院分区：
文献类型：
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作者：
Paix,Alexandre;Rasoloson,Dominique;Folkmann,Andrew;Seydoux,Geraldine
使用绿色荧光蛋白 (GFP) 等荧光报告基因标记蛋白质是确定蛋白质定位的有效方法，特别是在内源环境中标记蛋白质以保留天然基因组调控时。然而，将荧光报告基因插入哺乳动物细胞的基因组中需要构建含有选择标记和/或与插入位点同源的延伸序列（同源臂）的质粒。在这里，我们描述了一种简化的方案，该方案利用线性 DNA 的高倾向性，对 Cas9 RNA 引导的核酸内切酶诱导的 DNA 断裂进行同源定向修复，从而消除了所有克隆步骤。该方案使用具有短同源臂（30-40 bp）的 PCR 扩增子或合成基因片段，在特定基因组位置插入荧光报告基因。使用核转染或脂转染这两种转染程序之一将线性 DNA 引入具有预组装 Cas9-crRNA-tracrRNA 复合物的细胞中。该方案可在一周内完成，转染细胞的效率范围为 0.5% 至 20%，具体取决于目标位点。 © 2019 作者。
Tagging proteins with fluorescent reporters such as green fluorescent protein (GFP) is a powerful method to determine protein localization, especially when proteins are tagged in the endogenous context to preserve native genomic regulation. However, insertion of fluorescent reporters into the genomes of mammalian cells has required the construction of plasmids containing selection markers and/or extended sequences homologous to the site of insertion (homology arms). Here we describe a streamlined protocol that eliminates all cloning steps by taking advantage of the high propensity of linear DNAs to engage in homology‐directed repair of DNA breaks induced by the Cas9 RNA‐guided endonuclease. The protocol uses PCR amplicons, or synthetic gene fragments, with short homology arms (30‐40 bp) to insert fluorescent reporters at specific genomic locations. The linear DNAs are introduced into cells with preassembled Cas9‐crRNA‐tracrRNA complexes using one of two transfection procedures, nucleofection or lipofection. The protocol can be completed under a week, with efficiencies ranging from 0.5% to 20% of transfected cells depending on the locus targeted. © 2019 The Authors.