Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality
Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality
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DOI：
10.1016/j.molcel.2014.09.019
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发表时间：
2014-10-23
期刊：
影响因子：
16
通讯作者：
Barrangou, Rodolphe
中科院分区：
文献类型：
--
作者：
Briner, Alexandra E.;Donohoue, Paul D.;Barrangou, Rodolphe
RNA引导的Cas9内切核酸酶以序列依赖性方式特异性靶向并切割DNA，并已广泛用于可编程基因组编辑。Cas9活性依赖于与向导RNA的相互作用，并且进化上不同的Cas9核酸酶已显示正交地起作用。然而，选择性Cas9：向导RNA相互作用的分子基础知之甚少。在这里，我们鉴定并表征了天然crRNA中的六个保守模块：tracrRNA双链体和指导Cas9内切核酸酶活性的单向导RNA（sgRNA）。我们表明，凸起和连接是必要的DNA裂解，并表明，连接和发夹是有助于定义系统之间的正交性。相反，crRNA：tracrRNA互补区可以被修饰或部分去除。总的来说，我们的研究结果建立了引导RNA特征，这些特征驱动Cas9的DNA靶向，并为CRISPR技术开辟了新的设计和工程途径。
The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9: guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA: tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.