Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity
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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 journal › Journal article › Research › peer-review
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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