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 Cas9-sgRNA 复合物可保护脆弱的 DNA 限制性酶位点，使其免受克隆应用的切割


核酸酶死亡 Cas 蛋白 (dCas9) 的创建为大量新发现提供了一个新平台。多种 dCas9 工具已被开发用于转录调控、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，我们证明，dCas9和单向导RNA预组装形成核糖核蛋白dCas9-sgRNA（简称dRNP）能够特异性地、可逆地阻断限制性酶（RE）切割DNA的活性。我们发现，当 DNA 的识别或切割位点与 sgRNA 或原型间隔子相邻基序序列重叠时，RE 活性就会受到抑制。此外，我们发现多个 dRNP 可同时用于抑制多个 RE 位点。因此，我们利用这一新发现作为一种方法来证明插入片段可以连接到载体中，反之亦然，从而暂时隐藏选择性 RE 位点以允许所需的克隆。