Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Nierzwicki, Lukasz, East, Kyle W, Morzan, Uriel N, Arantes, Pablo R, Batista, Victor S, Lisi, George P, Palermo, Giulia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2021
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
_version_ 1784629434543767552
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
work_keys_str_mv AT nierzwickilukasz enhancedspecificitymutationsperturballostericsignalingincrisprcas9
AT eastkylew enhancedspecificitymutationsperturballostericsignalingincrisprcas9
AT morzanurieln enhancedspecificitymutationsperturballostericsignalingincrisprcas9
AT arantespablor enhancedspecificitymutationsperturballostericsignalingincrisprcas9
AT batistavictors enhancedspecificitymutationsperturballostericsignalingincrisprcas9
AT lisigeorgep enhancedspecificitymutationsperturballostericsignalingincrisprcas9
AT palermogiulia enhancedspecificitymutationsperturballostericsignalingincrisprcas9