The crystal structure of Cpf1 in complex with CRISPR RNA
The crystal structure of Cpf1 in complex with CRISPR RNA
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DOI：
10.1038/nature17944
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发表时间：
2016-04-28
期刊：
影响因子：
64.8
通讯作者：
Huang, Zhiwei
中科院分区：
文献类型：
--
作者：
Dong, De;Ren, Kuan;Huang, Zhiwei
以CRISPR-Cas9为例的CRISPR-Cas系统是一种rna引导的适应性免疫系统，被细菌和古细菌用来防御病毒感染（1-7）。CRISPR-Cpf1系统是一种新的2类CRISPR-Cas系统，在人类细胞中介导强大的DNA干扰（1,8-10）。尽管Cpf1和Cas9在功能上是保守的，但它们在许多方面存在差异，包括它们的引导rna和底物特异性。本文报道了结合CRISPR RNA （crRNA）的毛螺科细菌ND2006 Cpf1 （LbCpf1）的2.38 A晶体结构。LbCpf1具有三角形结构，中心有一个大的正电荷通道。crRNA被LbCpf1的寡核苷酸结合域识别，采用高度扭曲的构象，通过广泛的分子内相互作用和(Mg(H2O)(6))（2+）离子稳定。寡核苷酸结合结构域还包含一个环出螺旋结构域，这对LbCpf1底物结合很重要。结合crRNA或缺乏引导序列的crRNA会引起明显的构象变化，但不会引起LbCpf1的低聚化。我们的研究揭示了crRNA识别机制，揭示了crRNA引导的LbCpf1底物结合，建立了LbCpf1工程化的框架，提高了其基因组编辑的效率和特异性。
The CRISPR-Cas systems, as exemplified by CRISPR-Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection(1-7). The CRISPR-Cpf1 system, a new class 2 CRISPR-Cas system, mediates robust DNA interference in human cells(1,8-10). Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38 A crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H2O)(6))(2+) ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing.