programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
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发表时间：
2018
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通讯作者：
M. Jinek;Krzysztof Chylinski;Ines Fonfara;Michael Hauer;A. Jennifer;Doudna;Emmanuelle Charpentier
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作者：
M. Jinek;Krzysztof Chylinski;Ines Fonfara;Michael Hauer;A. Jennifer;Doudna;Emmanuelle Charpentier
CRISPR/Cas系统通过使用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。我们在这里展示了在这些系统的一个子集中，与反式激活tracrRNA碱基配对的成熟crRNA形成了一个双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-指导序列互补的位点处，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。
CRISPR/Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using crRNAs to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA base-paired to trans-activating 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 while the Cas9 RuvC-like domain cleaves the non-complementary 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.