Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA.
Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
Mol Cell. 2014 Oct 23;56(2):333-339. doi: 10.1016/j.molcel.2014.09.019. Epub 2014 Oct 16.
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.
RNA 引导的 Cas9 内切酶特异性地以序列依赖性方式靶向和切割 DNA，并已广泛用于可编程基因组编辑。Cas9 的活性依赖于与指导 RNA 的相互作用，并且已经证明进化上不同的 Cas9 核酸酶可以正交工作。然而，选择性 Cas9:guide-RNA 相互作用的分子基础理解甚少。在这里，我们在天然 crRNA:tracrRNA 双链体和单指导 RNA（sgRNA）中鉴定和表征了六个保守模块，这些模块指导 Cas9 内切酶活性。我们表明，凸起和连接体对于 DNA 切割是必需的，并证明连接体和发夹在系统之间的正交性定义中起着重要作用。相比之下，crRNA:tracrRNA 互补区可以被修饰或部分去除。总的来说，我们的结果确定了指导 RNA 的特征，这些特征驱动 Cas9 靶向 DNA，并为 CRISPR 技术开辟了新的设计和工程途径。
