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Structural insights into DNA cleavage activation of CRISPR-Cas9 system
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 d...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680257/ https://www.ncbi.nlm.nih.gov/pubmed/29123204 http://dx.doi.org/10.1038/s41467-017-01496-2 |
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author | Huai, Cong Li, Gan Yao, Ruijie Zhang, Yingyi Cao, Mi Kong, Liangliang Jia, Chenqiang Yuan, Hui Chen, Hongyan Lu, Daru Huang, Qiang |
author_facet | Huai, Cong Li, Gan Yao, Ruijie Zhang, Yingyi Cao, Mi Kong, Liangliang Jia, Chenqiang Yuan, Hui Chen, Hongyan Lu, Daru Huang, Qiang |
author_sort | Huai, Cong |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5680257 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-56802572017-11-15 Structural insights into DNA cleavage activation of CRISPR-Cas9 system Huai, Cong Li, Gan Yao, Ruijie Zhang, Yingyi Cao, Mi Kong, Liangliang Jia, Chenqiang Yuan, Hui Chen, Hongyan Lu, Daru Huang, Qiang Nat Commun Article 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. Nature Publishing Group UK 2017-11-09 /pmc/articles/PMC5680257/ /pubmed/29123204 http://dx.doi.org/10.1038/s41467-017-01496-2 Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Huai, Cong Li, Gan Yao, Ruijie Zhang, Yingyi Cao, Mi Kong, Liangliang Jia, Chenqiang Yuan, Hui Chen, Hongyan Lu, Daru Huang, Qiang Structural insights into DNA cleavage activation of CRISPR-Cas9 system |
title | Structural insights into DNA cleavage activation of CRISPR-Cas9 system |
title_full | Structural insights into DNA cleavage activation of CRISPR-Cas9 system |
title_fullStr | Structural insights into DNA cleavage activation of CRISPR-Cas9 system |
title_full_unstemmed | Structural insights into DNA cleavage activation of CRISPR-Cas9 system |
title_short | Structural insights into DNA cleavage activation of CRISPR-Cas9 system |
title_sort | structural insights into dna cleavage activation of crispr-cas9 system |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680257/ https://www.ncbi.nlm.nih.gov/pubmed/29123204 http://dx.doi.org/10.1038/s41467-017-01496-2 |
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