Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity

Research output: Contribution to journalJournal articlepeer-review

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Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity. / Stella, Stefano; Mesa, Pablo; Thomsen, Johannes; Paul, Bijoya; Alcón, Pablo; Jensen, Simon Bo; Saligram, Bhargav; Moses, Matias Emil; Hatzakis, Nikos S.; Montoya, Guillermo.

In: Cell, Vol. 175, No. 7, 13.12.2018, p. 1856-1871.e21.

Research output: Contribution to journalJournal articlepeer-review

Harvard

Stella, S, Mesa, P, Thomsen, J, Paul, B, Alcón, P, Jensen, SB, Saligram, B, Moses, ME, Hatzakis, NS & Montoya, G 2018, 'Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity', Cell, vol. 175, no. 7, pp. 1856-1871.e21. https://doi.org/10.1016/j.cell.2018.10.045

APA

Stella, S., Mesa, P., Thomsen, J., Paul, B., Alcón, P., Jensen, S. B., Saligram, B., Moses, M. E., Hatzakis, N. S., & Montoya, G. (2018). Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity. Cell, 175(7), 1856-1871.e21. https://doi.org/10.1016/j.cell.2018.10.045

Vancouver

Stella S, Mesa P, Thomsen J, Paul B, Alcón P, Jensen SB et al. Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity. Cell. 2018 Dec 13;175(7):1856-1871.e21. https://doi.org/10.1016/j.cell.2018.10.045

Author

Stella, Stefano ; Mesa, Pablo ; Thomsen, Johannes ; Paul, Bijoya ; Alcón, Pablo ; Jensen, Simon Bo ; Saligram, Bhargav ; Moses, Matias Emil ; Hatzakis, Nikos S. ; Montoya, Guillermo. / Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity. In: Cell. 2018 ; Vol. 175, No. 7. pp. 1856-1871.e21.

Bibtex

@article{339c436d17fd4d908fcb98cf47baa702,
title = "Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity",
abstract = "Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.",
author = "Stefano Stella and Pablo Mesa and Johannes Thomsen and Bijoya Paul and Pablo Alc{\'o}n and Jensen, {Simon Bo} and Bhargav Saligram and Moses, {Matias Emil} and Hatzakis, {Nikos S.} and Guillermo Montoya",
note = "Copyright {\textcopyright} 2018 Elsevier Inc. All rights reserved.",
year = "2018",
month = dec,
day = "13",
doi = "10.1016/j.cell.2018.10.045",
language = "English",
volume = "175",
pages = "1856--1871.e21",
journal = "Cell",
issn = "0092-8674",
publisher = "Cell Press",
number = "7",

}

RIS

TY - JOUR

T1 - Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity

AU - Stella, Stefano

AU - Mesa, Pablo

AU - Thomsen, Johannes

AU - Paul, Bijoya

AU - Alcón, Pablo

AU - Jensen, Simon Bo

AU - Saligram, Bhargav

AU - Moses, Matias Emil

AU - Hatzakis, Nikos S.

AU - Montoya, Guillermo

N1 - Copyright © 2018 Elsevier Inc. All rights reserved.

PY - 2018/12/13

Y1 - 2018/12/13

N2 - Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.

AB - Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.

U2 - 10.1016/j.cell.2018.10.045

DO - 10.1016/j.cell.2018.10.045

M3 - Journal article

C2 - 30503205

VL - 175

SP - 1856-1871.e21

JO - Cell

JF - Cell

SN - 0092-8674

IS - 7

ER -

ID: 210196522