The creation of the nuclease-dead Cas protein (dCas9) offers a new platform for a plethora of new discoveries. Diverse dCas9 tools have been developed for transcription regulation, epigenetic engineering, base editing, genome imaging, genetic screens, and chromatin immunoprecipitation. Here, we show that dCas9 and single-guide RNA preassembled to form ribonucleoprotein dCas9–sgRNA (referred to as dRNP) is capable of specifically and reversibly blocking the activity of DNA cleavage by restriction enzymes (REs). We show that the inhibition of RE activities occurs when the recognition or the cleavage site of the DNA is overlapped by the sgRNA or the protospacer adjacent motif sequence. Furthermore, we show that multiple dRNPs can be used simultaneously to inhibit more than one RE sites. As such, we exploited this novel finding as a method to demonstrate that inserts can be ligated into vectors, and vice versa, whereby selective RE sites are temporarily sheltered to allow the desired cloning.

中文翻译：

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死的 Cas9-sgRNA 复合物从裂解中保护脆弱的 DNA 限制酶位点，用于克隆应用

核酸酶死亡 Cas 蛋白 (dCas9) 的产生为大量新发现提供了一个新平台。已经开发了多种 dCas9 工具用于转录调控、表观遗传工程、碱基编辑、基因组成像、遗传筛选和染色质免疫沉淀。在这里，我们表明 dCas9 和单向导 RNA 预组装形成核糖核蛋白 dCas9-sgRNA（称为 dRNP）能够特异性和可逆地阻断限制性酶（RE）对 DNA 切割的活性。我们表明，当 DNA 的识别或切割位点与 sgRNA 或原型间隔区相邻基序序列重叠时，就会发生 RE 活性的抑制。此外，我们表明可以同时使用多个 dRNP 来抑制多个 RE 位点。像这样，反之亦然，从而选择性的 RE 位点被暂时庇护以允许所需的克隆。