A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
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DOI：
10.1126/science.1225829
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发表时间：
2012-08-17
期刊：
影响因子：
56.9
通讯作者：
Charpentier, Emmanuelle
中科院分区：
文献类型：
--
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
规则间隔短回文重复序列（CRISPR）/CRISPR相关（Cas）系统通过使用CRISPR RNA（crRNA）来引导入侵核酸的沉默，为细菌和古细菌提供针对病毒和质粒的适应性免疫。我们在此显示，在这些系统的一个子集中，与反式激活crRNA（tracrRNA）碱基配对的成熟crRNA形成双RNA结构，该结构指导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，当被工程化为单个RNA嵌合体时，也指导序列特异性Cas9 dsDNA切割。我们的研究揭示了一个使用双RNA进行位点特异性DNA切割的内切核酸酶家族，并强调了开发该系统进行RNA可编程基因组编辑的潜力。
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.