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Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy
The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9–RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize...
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/PMC5681550/ https://www.ncbi.nlm.nih.gov/pubmed/29127285 http://dx.doi.org/10.1038/s41467-017-01466-8 |
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author | Shibata, Mikihiro Nishimasu, Hiroshi Kodera, Noriyuki Hirano, Seiichi Ando, Toshio Uchihashi, Takayuki Nureki, Osamu |
author_facet | Shibata, Mikihiro Nishimasu, Hiroshi Kodera, Noriyuki Hirano, Seiichi Ando, Toshio Uchihashi, Takayuki Nureki, Osamu |
author_sort | Shibata, Mikihiro |
collection | PubMed |
description | The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9–RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9–RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9. |
format | Online Article Text |
id | pubmed-5681550 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-56815502017-11-16 Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy Shibata, Mikihiro Nishimasu, Hiroshi Kodera, Noriyuki Hirano, Seiichi Ando, Toshio Uchihashi, Takayuki Nureki, Osamu Nat Commun Article The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9–RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9–RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9. Nature Publishing Group UK 2017-11-10 /pmc/articles/PMC5681550/ /pubmed/29127285 http://dx.doi.org/10.1038/s41467-017-01466-8 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 Shibata, Mikihiro Nishimasu, Hiroshi Kodera, Noriyuki Hirano, Seiichi Ando, Toshio Uchihashi, Takayuki Nureki, Osamu Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy |
title | Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy |
title_full | Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy |
title_fullStr | Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy |
title_full_unstemmed | Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy |
title_short | Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy |
title_sort | real-space and real-time dynamics of crispr-cas9 visualized by high-speed atomic force microscopy |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681550/ https://www.ncbi.nlm.nih.gov/pubmed/29127285 http://dx.doi.org/10.1038/s41467-017-01466-8 |
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