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Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9
CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8741213/ https://www.ncbi.nlm.nih.gov/pubmed/34908530 http://dx.doi.org/10.7554/eLife.73601 |
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author | Nierzwicki, Lukasz East, Kyle W Morzan, Uriel N Arantes, Pablo R Batista, Victor S Lisi, George P Palermo, Giulia |
author_facet | Nierzwicki, Lukasz East, Kyle W Morzan, Uriel N Arantes, Pablo R Batista, Victor S Lisi, George P Palermo, Giulia |
author_sort | Nierzwicki, Lukasz |
collection | PubMed |
description | CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system’s function. |
format | Online Article Text |
id | pubmed-8741213 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-87412132022-01-11 Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9 Nierzwicki, Lukasz East, Kyle W Morzan, Uriel N Arantes, Pablo R Batista, Victor S Lisi, George P Palermo, Giulia eLife Structural Biology and Molecular Biophysics CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system’s function. eLife Sciences Publications, Ltd 2021-12-15 /pmc/articles/PMC8741213/ /pubmed/34908530 http://dx.doi.org/10.7554/eLife.73601 Text en © 2021, Nierzwicki et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Structural Biology and Molecular Biophysics Nierzwicki, Lukasz East, Kyle W Morzan, Uriel N Arantes, Pablo R Batista, Victor S Lisi, George P Palermo, Giulia Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9 |
title | Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9 |
title_full | Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9 |
title_fullStr | Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9 |
title_full_unstemmed | Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9 |
title_short | Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9 |
title_sort | enhanced specificity mutations perturb allosteric signaling in crispr-cas9 |
topic | Structural Biology and Molecular Biophysics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8741213/ https://www.ncbi.nlm.nih.gov/pubmed/34908530 http://dx.doi.org/10.7554/eLife.73601 |
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