College of Health and Life Sciences, Division of Biological and Biomedical Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha, Qatar.
CRISPR J. 2021 Apr;4(2):275-289. doi: 10.1089/crispr.2020.0134.
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 , whereby selective RE sites are temporarily sheltered to allow the desired cloning.
核酸酶失活 Cas 蛋白（dCas9）的创建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、表观遗传学工程、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 和单指导 RNA 预先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酶（REs）对 DNA 切割的活性。我们表明，当 sgRNA 或前间隔基序序列与 DNA 的识别或切割位点重叠时，RE 活性会受到抑制。此外，我们表明可以同时使用多个 dRNPs 来抑制多个 RE 位点。因此，我们利用这一新发现作为一种方法来证明可以将插入物连接到载体中，并且可以选择性地屏蔽特定的 RE 位点以允许所需的克隆。
