Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032, Japan; JST, PRESTO, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032, Japan.
Broad Institute of MIT and Harvard, Cambridge, MA 02141, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13.
The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.
CRISPR 相关内切酶 Cas9 可通过单链向导 RNA（sgRNA）靶向特定的基因组位点。在此，我们报告了酿脓链球菌 Cas9 与 sgRNA 及其靶 DNA 复合物的晶体结构，分辨率为 2.5 Å。该结构揭示了一种由目标识别和核酸酶叶瓣组成的双叶结构，sgRNA:DNA 异源双链体容纳在其界面的正电荷沟道中。虽然识别叶瓣对于结合 sgRNA 和 DNA 是必不可少的，但核酸酶叶瓣包含 HNH 和 RuvC 核酸酶结构域，它们分别位于切割靶 DNA 互补和非互补链的适当位置。核酸酶叶瓣还包含一个羧基末端结构域，负责与间隔相邻基序（PAM）相互作用。这个高分辨率结构和伴随的功能分析揭示了 Cas9 通过 RNA 引导的 DNA 靶向的分子机制，从而为新的、多功能基因组编辑技术的合理设计铺平了道路。
