C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism
C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism
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C2c1-sgRNA 复合物结构揭示了 RNA 引导的 DNA 切割机制
DOI：
10.1016/j.molcel.2016.11.040
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发表时间：
2017-01-19
期刊：
影响因子：
16
通讯作者：
Wang, Yanli
中科院分区：
文献类型：
--
作者：
Liu, Liang;Chen, Peng;Wang, Yanli
C2c1是一种V - B型CRISPR - Cas系统双RNA引导的DNA内切核酸酶。在此，我们报道了嗜酸热脂环酸芽孢杆菌（Alicyclobacillus acidoterrestris）C2c1与嵌合单分子向导RNA（sgRNA）复合物的晶体结构。嗜酸热脂环酸芽孢杆菌C2c1（AacC2c1）呈现出双叶结构，由REC叶和NUC叶组成。sgRNA支架形成一种四螺旋结构，与先前的预测不同。crRNA位于C2c1的中央通道，而tracrRNA位于外表面凹槽中。尽管AacC2c1缺乏前间隔序列邻近基序（PAM）相互作用结构域，但我们的分析显示，PAM双链体的结合位置与Cpf1中的类似。重要的是，C2c1 - sgRNA系统对向导RNA与靶DNA之间的单核苷酸错配高度敏感。由此导致的脱靶切割减少，使C2c1成为当前基因组编辑工具库中有价值的新成员。总之，我们的研究结果表明，sgRNA组装是通过一种与先前报道的Cas9或Cpf1内切核酸酶不同的机制实现的。
C2c1 is a type V-B CRISPR-Cas system dual-RNAguided DNA endonuclease. Here, we report the crystal structure of Alicyclobacillus acidoterrestris C2c1 in complex with a chimeric single-molecule guide RNA ( sgRNA). AacC2c1 exhibits a bi-lobed architecture consisting of a REC and NUC lobe. The sgRNA scaffold forms a tetra-helical structure, distinct from previous predictions. The crRNA is located in the central channel of C2c1, and the tracrRNA resides in an external surface groove. Although AacC2c1 lacks a PAM-interacting domain, our analysis revealed that the PAM duplex has a similar binding position found in Cpf1. Importantly, C2c1-sgRNA system is highly sensitive to singlenucleotide mismatches between guide RNA and target DNA. The resulting reduction in off-target cleavage renders C2c1 a valuable addition to the current arsenal of genome-editing tools. Together, our findings indicate that sgRNA assembly is achieved through a mechanism distinct from that reported previously for Cas9 or Cpf1 endonucleases.