Structural insights into DNA cleavage activation of CRISPR-Cas9 system

• Published in: Nature communications Vol. 8; no. 1; pp. 1375 - 9
• Main Authors: Huai, Cong, Li, Gan, Yao, Ruijie, Zhang, Yingyi, Cao, Mi, Kong, Liangliang, Jia, Chenqiang, Yuan, Hui, Chen, Hongyan, Lu, Daru, Huang, Qiang
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 09.11.2017; Nature Publishing Group; Nature Portfolio
• Subjects: 631/208/4041; 631/45/173; 631/535/1258/1259; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cleavage; CRISPR; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Cryoelectron Microscopy; Deoxyribonucleic acid; DNA; DNA Cleavage; DNA structure; Endonuclease; Endonucleases - chemistry; Endonucleases - genetics; Endonucleases - metabolism; Genome editing; Genomes; Humanities and Social Sciences; Molecular dynamics; Molecular Dynamics Simulation; multidisciplinary; Mutation; Nuclease; Protein Domains; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - chemistry; RNA, Guide, CRISPR-Cas Systems - metabolism; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-017-01496-2

CRISPR-Cas9 technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas9 binds specifically to target DNA and then cleaves the two DNA strands with HNH and RuvC nuclease domains. However, structural information regarding the DNA cleavage-activating state of two nuclease domains remains sparse. Here, we report a 5.2 Å cryo-EM structure of Cas9 in complex with sgRNA and target DNA. This structure reveals a conformational state of Cas9 in which the HNH domain is closest to the DNA cleavage site. Compared with two known HNH states, our structure shows that the HNH active site moves toward the cleavage site by about 25 and 13 Å, respectively. In combination with EM-based molecular dynamics simulations, we show that residues of the nuclease domains in our structure could form cleavage-compatible conformations with the target DNA. Together, these results strongly suggest that our cryo-EM structure resembles a DNA cleavage-activating architecture of Cas9. CRISPR-Cas9 is widely used for genome engineering but structural data for the DNA cleavage step are still incomplete. Here, the authors present the cryo-EM structure of a ternary Cas9-sgRNA-target DNA complex, perform MD simulations and discuss implications for the Cas9 DNA cleavage mechanism.