Mol Cell. 2019 Dec 19;76(6):938-952.e5.

Wei Sun ¹ , Jing Yang ² , Zhi Cheng ³ , Nadia Amrani ⁴ , Chao Liu ³ , Kangkang Wang ³ , Raed Ibraheim ⁴ , Alireza Edraki ⁴ , Xue Huang ⁵ , Min Wang ¹ , Jiuyu Wang ¹ , Liang Liu ¹ , Gang Sheng ¹ , Yanhua Yang ⁶ , Jizhong Lou ⁷ , Erik J Sontheimer ⁸ , Yanli Wang ⁹

1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
2. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China.
3. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
4. RNA Therapeutics Institute, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605, USA.
5. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Hefei National Research Center for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China.
6. Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China.
7. Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
8. RNA Therapeutics Institute, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605, USA. Electronic address: erik.sontheimer@umassmed.edu.
9. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Collaborative Innovation Center of Genetics and Development, Shanghai 200438, China. Electronic address: ylwang@ibp.ac.cn.

Abstract

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-DNA-bound state. In the HNH catalytic state of Nme1Cas9, 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, guide-target engagement, cleavage mechanism, and anti-CRISPR inhibition, facilitating the optimization of these genome-editing platforms.

Presented by Wei Sun