Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States
Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States
复制标题
催化平衡和抗 CRISPR 抑制状态下脑膜炎奈瑟菌 Cas9 复合物的结构
DOI：
10.1016/j.molcel.2019.09.025
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发表时间：
2019-12-19
期刊：
影响因子：
16
通讯作者：
Wang, Yanli
中科院分区：
文献类型：
--
作者：
Sun, Wei;Yang, Jing;Wang, Yanli
高分辨率的Cas9结构尚未揭示催化构象，因为HNH核酸酶结构域位于远离裂解位点的位置。Nme1Cas9和Nme2Cas9是体内基因组编辑的紧凑型核酸酶。在这里，我们报道了脑膜炎球菌Cas9同源物与sgRNA、dsDNA或AcrIIC3抗crispr蛋白复合物的结构。dna结合结构代表了目标识别的早期步骤，随后的HNH预催化状态，HNH催化状态和切割目标- dnabound状态。在Nmel Cas9的HNH催化状态下，活性位点位于目标链的可剪切磷酸二酯链上，提供了活性构象的高分辨率视图。HNH活性构象激活RuvC结构域。我们的结构解释了Nme1Cas9和Nme2Cas9如何读取不同的PAM序列，以及AcrIIC3如何抑制Nme1Cas9的活性。这些结构提供了对Cas9结构域重排、引导靶标接合、切割机制和抗crispr抑制的见解，促进了这些基因组编辑平台的优化。
High-resolution Cas9 structures have yet to reveal catalytic conformations due to HNH nuclease domain positioning away from the cleavage site. Nme1Cas9 and Nme2Cas9 are compact nucleases for in vivo genome editing. Here, we report structures of meningococcal Cas9 homologs in complex with sgRNA, dsDNA, or the AcrIIC3 anti-CRISPR protein. DNA-bound structures represent an early step of target recognition, a later HNH pre-catalytic state, the HNH catalytic state, and a cleaved-target-DNAbound state. In the HNH catalytic state of Nmel Cas9, the active site is seen poised at the scissile phosphodiester linkage of the target strand, providing a high-resolution view of the active conformation. The HNH active conformation activates the RuvC domain. Our structures explain how Nme1Cas9 and Nme2Cas9 read distinct PAM sequences and how AcrIIC3 inhibits Nme1Cas9 activity. These structures provide insights into Cas9 domain rearrangements, guidetarget engagement, cleavage mechanism, and anti-CRISPR inhibition, facilitating the optimization of these genome-editing platforms.