天然脑膜炎奈瑟菌 CRISPR-Cas9 系统对 RNA 的可编程切割和识别。
Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis.
机构信息
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
出版信息
Mol Cell. 2018 Mar 1;69(5):906-914.e4. doi: 10.1016/j.molcel.2018.01.025. Epub 2018 Feb 15.
The microbial CRISPR systems enable adaptive defense against mobile elements and also provide formidable tools for genome engineering. The Cas9 proteins are type II CRISPR-associated, RNA-guided DNA endonucleases that identify double-stranded DNA targets by sequence complementarity and protospacer adjacent motif (PAM) recognition. Here we report that the type II-C CRISPR-Cas9 from Neisseria meningitidis (Nme) is capable of programmable, RNA-guided, site-specific cleavage and recognition of single-stranded RNA targets and that this ribonuclease activity is independent of the PAM sequence. We define the mechanistic feature and specificity constraint for RNA cleavage by NmeCas9 and also show that nuclease null dNmeCas9 binds to RNA target complementary to CRISPR RNA. Finally, we demonstrate that NmeCas9-catalyzed RNA cleavage can be blocked by three families of type II-C anti-CRISPR proteins. These results fundamentally expand the targeting capacities of CRISPR-Cas9 and highlight the potential utility of NmeCas9 as a single platform to target both RNA and DNA.
微生物的 CRISPR 系统能够实现对移动元件的适应性防御，同时也为基因组工程提供了强大的工具。Cas9 蛋白是 II 型 CRISPR 相关的、RNA 指导的 DNA 内切酶，通过序列互补性和原间隔序列邻近基序（PAM）识别来识别双链 DNA 靶标。在这里，我们报告称脑膜炎奈瑟菌（Nme）的 II 型-C CRISPR-Cas9 能够对单链 RNA 靶标进行可编程、RNA 指导的、位点特异性切割和识别，并且这种核糖核酸酶活性与 PAM 序列无关。我们定义了 NmeCas9 的 RNA 切割的机制特征和特异性限制，还表明无核酸酶的 dNmeCas9 与与 CRISPR RNA 互补的 RNA 靶标结合。最后，我们证明 NmeCas9 催化的 RNA 切割可以被三种类型 II-C 抗 CRISPR 蛋白家族阻断。这些结果从根本上扩展了 CRISPR-Cas9 的靶向能力，并强调了 NmeCas9 作为一个单一平台同时靶向 RNA 和 DNA 的潜在用途。
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