Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
Science. 2012 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829. Epub 2012 Jun 28.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
成簇规律间隔短回文重复序列 (CRISPR)/CRISPR 相关 (Cas) 系统通过使用 CRISPR RNA (crRNA) 引导沉默入侵核酸，为细菌和古菌提供了针对病毒和质粒的适应性免疫。我们在这里表明，在这些系统的一部分中，与转激活 crRNA (tracrRNA) 碱基配对的成熟 crRNA 形成了一种双链 RNA 结构，指导 CRISPR 相关蛋白 Cas9 在靶 DNA 中引入双链 (ds) 断裂。在与 crRNA 引导序列互补的位点，Cas9 HNH 核酸酶结构域切割互补链，而 Cas9 RuvC 样结构域切割非互补链。双 tracrRNA:crRNA ，当作为单个 RNA 嵌合体进行工程设计时，也可指导序列特异性 Cas9 dsDNA 切割。我们的研究揭示了一类使用双链 RNA 进行位点特异性 DNA 切割的内切酶，并强调了利用该系统进行 RNA 可编程基因组编辑的潜力。
