Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9.
Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9.
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DOI：
10.1002/cpmb.100
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发表时间：
2019-09-01
影响因子：
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通讯作者：
Sheltzer, Jason M
中科院分区：
文献类型：
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作者：
Giuliano, Christopher J;Lin, Ann;Sheltzer, Jason M
CRISPR/Cas9技术能够在哺乳动物细胞中的靶基因中快速产生功能丧失突变。携带这些突变的单个细胞可用于建立新的细胞系，从而创建CRISPR诱导的敲除克隆。这些克隆细胞系是探索蛋白质功能、分析基因丢失后果和研究生物试剂特异性的重要工具。然而，敲除克隆的成功衍生可能在技术上具有挑战性，并且可能因多种因素而复杂化，包括不完全的靶消融和克隆间异质性。在这里，我们描述了优化的协议和质粒产生克隆敲除哺乳动物细胞系。我们提供了指导RNA设计，CRISPR递送和敲除验证的策略，这些策略有助于获得真正的敲除克隆，并适合于多重基因靶向。这些方案将广泛适用于寻求应用CRISPR研究哺乳动物细胞中基因功能的研究人员。版权所有2019作者。
CRISPR/Cas9 technology enables the rapid generation of loss-of-function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. © 2019 The Authors.