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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...

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Autores principales: Huai, Cong, Li, Gan, Yao, Ruijie, Zhang, Yingyi, Cao, Mi, Kong, Liangliang, Jia, Chenqiang, Yuan, Hui, Chen, Hongyan, Lu, Daru, Huang, Qiang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
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.
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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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