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Roadmap for the use of base editors to decipher drug mechanism of action
- Source :
- Dipòsit Digital de la UB, Universidad de Barcelona, PLoS ONE, Vol 16, Iss 9, p e0257537 (2021), PLoS ONE
- Publication Year :
- 2021
- Publisher :
- Public Library of Science (PLoS), 2021.
-
Abstract
- Background: 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, and we focus on G-protein coupled receptors.Results: We set up a pooled mutagenesis screening framework 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. We thus target the Beta 2 Adrenergic Receptor (B2AR) and employ a 6xCRE-mCherry reporter system to monitor its activity. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we then 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. Conclusions: The base editing technology has a great potential to help deciphering the mechanism of action of drugs, and it is a very powerful tool in drug discovery. Here we show an application of pooled mutagenesis screening to study B2AR, and we provide a roadmap for successfully applying this approach to other target proteins.
- Subjects :
- Computer science
Biochemistry
Genes, Reporter
Medicine and Health Sciences
CRISPR
RNA, Small Interfering
Investigació farmacèutica
Subgenomic mRNA
Gene Editing
Multidisciplinary
Drug discovery
Drugs
Genomics
Small interfering RNA
Nucleic acids
Mutagènesi
Medicine
DECIPHER
RNA Interference
medicine.symptom
Research Article
Signal Transduction
RNA, Guide, Kinetoplastida
Pharmaceutical research
Transmembrane Receptors
Science
Mutagenesis (molecular biology technique)
Context (language use)
Computational biology
Transfection
Research and Analysis Methods
Green Fluorescent Protein
Genetics
medicine
Humans
Molecular Biology Techniques
Non-coding RNA
Molecular Biology
Pharmacology
Isoproterenol
Biology and Life Sciences
Proteins
Cell Biology
Gene regulation
Luminescent Proteins
HEK293 Cells
Mechanism of action
Mutagenesis
Mutagenesis, Site-Directed
RNA
Gene expression
Receptors, Adrenergic, beta-2
CRISPR-Cas Systems
G Protein Coupled Receptors
Cloning
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Dipòsit Digital de la UB, Universidad de Barcelona, PLoS ONE, Vol 16, Iss 9, p e0257537 (2021), PLoS ONE
- Accession number :
- edsair.doi.dedup.....a0c0ca6f9b0b2575d8c0a2a63639f238