将 CRISPR 重新用作 RNA 引导的平台，用于基因表达的序列特异性控制。
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.
机构信息
UCSF Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
出版信息
Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
在全基因组范围内进行靶向基因调控是一种强大的策略，可以用于研究、干扰和工程细胞系统。在这里，我们开发了一种基于 Cas9 的基因表达控制方法，Cas9 是一种来自 II 型 CRISPR 系统的 RNA 指导的 DNA 内切酶。我们表明，一种缺乏内切酶活性的无活性 Cas9，与指导 RNA 共表达时，会产生一种 DNA 识别复合物，该复合物可以特异性地干扰转录延伸、RNA 聚合酶结合或转录因子结合。我们将这种系统称为 CRISPR 干扰（CRISPRi），它可以有效地抑制大肠杆菌中靶向基因的表达，并且没有可检测的脱靶效应。CRISPRi 可用于同时抑制多个靶基因，其效果是可逆的。我们还提供了证据表明，该系统可以适应哺乳动物细胞中的基因抑制。这种 RNA 指导的 DNA 识别平台为在全基因组范围内选择性干扰基因表达提供了一种简单的方法。
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