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Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen‐Deuterium Exchange Mass Spectrometry. / Zhdanova, Polina V.; Chernonosov, Alexander A.; Prokhorova, Daria V. и др.

в: International Journal of Molecular Sciences, Том 23, № 3, 1129, 01.02.2022.

Результаты исследований: Научные публикации в периодических изданияхстатьяРецензирование

Harvard

Zhdanova, PV, Chernonosov, AA, Prokhorova, DV, Stepanov, GA, Kanazhevskaya, LY & Koval, VV 2022, 'Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen‐Deuterium Exchange Mass Spectrometry', International Journal of Molecular Sciences, Том. 23, № 3, 1129. https://doi.org/10.3390/ijms23031129

APA

Zhdanova, P. V., Chernonosov, A. A., Prokhorova, D. V., Stepanov, G. A., Kanazhevskaya, L. Y., & Koval, V. V. (2022). Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen‐Deuterium Exchange Mass Spectrometry. International Journal of Molecular Sciences, 23(3), [1129]. https://doi.org/10.3390/ijms23031129

Vancouver

Zhdanova PV, Chernonosov AA, Prokhorova DV, Stepanov GA, Kanazhevskaya LY, Koval VV. Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen‐Deuterium Exchange Mass Spectrometry. International Journal of Molecular Sciences. 2022 февр. 1;23(3):1129. doi: 10.3390/ijms23031129

Author

Zhdanova, Polina V. ; Chernonosov, Alexander A. ; Prokhorova, Daria V. и др. / Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen‐Deuterium Exchange Mass Spectrometry. в: International Journal of Molecular Sciences. 2022 ; Том 23, № 3.

BibTeX

@article{733df17bb4904253895c34e49931e8e3,
title = "Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen‐Deuterium Exchange Mass Spectrometry",
abstract = "The Cas9 endonuclease is an essential component of the CRISPR–Cas‐based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR–Cas9 system. A deep under-standing of the Cas9 mechanism and its structural‐functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX‐MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to un-dergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX‐MS for the elucidation of mechanistic aspects of Cas9 functioning.",
keywords = "Cas9, CRISPR–Cas systems, HDX‐MS, Hydrogen‐deuterium exchange mass spectrometry, Molecular dynamics, Single guide RNA",
author = "Zhdanova, {Polina V.} and Chernonosov, {Alexander A.} and Prokhorova, {Daria V.} and Stepanov, {Grigory A.} and Kanazhevskaya, {Lyubov Yu} and Koval, {Vladimir V.}",
note = "Funding Information: Funding: This research was supported by the Russian Science Foundation (grant no. 20‐14‐00214). Publisher Copyright: {\textcopyright} 2022 by the authors. Licensee MDPI, Basel, Switzerland.",
year = "2022",
month = feb,
day = "1",
doi = "10.3390/ijms23031129",
language = "English",
volume = "23",
journal = "International Journal of Molecular Sciences",
issn = "1661-6596",
publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",
number = "3",

}

RIS

TY - JOUR

T1 - Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen‐Deuterium Exchange Mass Spectrometry

AU - Zhdanova, Polina V.

AU - Chernonosov, Alexander A.

AU - Prokhorova, Daria V.

AU - Stepanov, Grigory A.

AU - Kanazhevskaya, Lyubov Yu

AU - Koval, Vladimir V.

N1 - Funding Information: Funding: This research was supported by the Russian Science Foundation (grant no. 20‐14‐00214). Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2022/2/1

Y1 - 2022/2/1

N2 - The Cas9 endonuclease is an essential component of the CRISPR–Cas‐based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR–Cas9 system. A deep under-standing of the Cas9 mechanism and its structural‐functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX‐MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to un-dergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX‐MS for the elucidation of mechanistic aspects of Cas9 functioning.

AB - The Cas9 endonuclease is an essential component of the CRISPR–Cas‐based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR–Cas9 system. A deep under-standing of the Cas9 mechanism and its structural‐functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX‐MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to un-dergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX‐MS for the elucidation of mechanistic aspects of Cas9 functioning.

KW - Cas9

KW - CRISPR–Cas systems

KW - HDX‐MS

KW - Hydrogen‐deuterium exchange mass spectrometry

KW - Molecular dynamics

KW - Single guide RNA

UR - http://www.scopus.com/inward/record.url?scp=85122943864&partnerID=8YFLogxK

U2 - 10.3390/ijms23031129

DO - 10.3390/ijms23031129

M3 - Article

C2 - 35163047

AN - SCOPUS:85122943864

VL - 23

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 3

M1 - 1129

ER -

ID: 35322735