Roadmap for the use of base editors to decipher drug mechanism of action.

CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context focusing on G-protein coupled recepto...

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Autores principales: Estel Aparicio-Prat, Dong Yan, Marco Mariotti, Michael Bassik, Gaelen Hess, Jean-Philippe Fortin, Andrea Weston, Hualin S Xi, Robert Stanton
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Publicado: Public Library of Science (PLoS) 2021
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Acceso en línea:https://doaj.org/article/7653c551366442d8a8e4bbbeede6a9dd
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spelling oai:doaj.org-article:7653c551366442d8a8e4bbbeede6a9dd2021-12-02T20:14:19ZRoadmap for the use of base editors to decipher drug mechanism of action.1932-620310.1371/journal.pone.0257537https://doaj.org/article/7653c551366442d8a8e4bbbeede6a9dd2021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0257537https://doaj.org/toc/1932-6203CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context focusing on G-protein coupled receptors. A pooled mutagenesis screening framework was set up based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. The β2 Adrenergic Receptor (B2AR) was targeted in this way employing a 6xCRE-mCherry reporter system to monitor its response to isoproterenol. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology.Estel Aparicio-PratDong YanMarco MariottiMichael BassikGaelen HessJean-Philippe FortinAndrea WestonHualin S XiRobert StantonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 9, p e0257537 (2021)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Estel Aparicio-Prat
Dong Yan
Marco Mariotti
Michael Bassik
Gaelen Hess
Jean-Philippe Fortin
Andrea Weston
Hualin S Xi
Robert Stanton
Roadmap for the use of base editors to decipher drug mechanism of action.
description CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context focusing on G-protein coupled receptors. A pooled mutagenesis screening framework was set up based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. The β2 Adrenergic Receptor (B2AR) was targeted in this way employing a 6xCRE-mCherry reporter system to monitor its response to isoproterenol. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology.
format article
author Estel Aparicio-Prat
Dong Yan
Marco Mariotti
Michael Bassik
Gaelen Hess
Jean-Philippe Fortin
Andrea Weston
Hualin S Xi
Robert Stanton
author_facet Estel Aparicio-Prat
Dong Yan
Marco Mariotti
Michael Bassik
Gaelen Hess
Jean-Philippe Fortin
Andrea Weston
Hualin S Xi
Robert Stanton
author_sort Estel Aparicio-Prat
title Roadmap for the use of base editors to decipher drug mechanism of action.
title_short Roadmap for the use of base editors to decipher drug mechanism of action.
title_full Roadmap for the use of base editors to decipher drug mechanism of action.
title_fullStr Roadmap for the use of base editors to decipher drug mechanism of action.
title_full_unstemmed Roadmap for the use of base editors to decipher drug mechanism of action.
title_sort roadmap for the use of base editors to decipher drug mechanism of action.
publisher Public Library of Science (PLoS)
publishDate 2021
url https://doaj.org/article/7653c551366442d8a8e4bbbeede6a9dd
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