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Roadmap for the use of Base Editors to Decipher Drug Mechanism of Action

Estel Aparicio-Prat, Dong Yan, Marco Mariotti, Michael Bassik, Gaelen Hess, Jean-Philippe Fortin, Andrea Weston, Hualin S. Xi, Robert Stanton

Abstract
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.

Introduction
The use of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) with Cas9 (CRISPR associated protein 9) has proven to be a revolutionary technology enabling the rapid introduction of genetic perturbations at targeted genomic regions. Initially, CRISPR was primarily used as a tool for specific and efficient gene knockout.

Materials and methods
Cell lines

Transfection was performed with Lipofectamine 2000 (ThermoFisher) or Lipofectamine CRISPRMAX Cas9 Transfection reagent (ThermoFisher). Electroporation was performed using Neon transfection system (ThermoFisher).

Bioinformatic analysis
BCL files from the sequencing were converted to FASTQ format using BCL2FASTQ (Illumina). SamTools was then used for alignment to the amplicon sequence using a quality score of 30. Mutagenesis rate per position in the amplicon was calculated as “reads of non-reference base allele/total reads”. Mutagenesis rate of 1 is the maximum and means all alleles have been edited. We normalized the mutagenesis values per sample obtaining mutagenesis z-scores per genomic position, and we computed the difference of z-scores between the mCherry negative sorted sample compared to the presorted sample.

Discussion
The objective of this study was to evaluate the utility of base editors in early drug discovery, as a tool for understanding mechanisms of compound pharmacology and target biology. This has been traditionally done through alanine scanning and site directed mutagenesis, however the development of CRISPR-based gene editing has created the opportunity for a less time-consuming and more high-throughput approach. After designing an effective reporter system, the delivery a pool of sgRNA at a low MOI followed by flow cytometry allows the separation of cells into responding and non-responding cohorts, and analysis of sequencing data pinpoints to those mutations with functional effects.

Acknowledgments: We thank Hanna Sobon from the Flow Cytometry facility and Justin Boyd from the High Content facility at Pfizer Cambridge for their support.

Citation: Aparicio-Prat E, Yan D, Mariotti M, Bassik M, Hess G, Fortin J-P, et al. (2021) Roadmap for the use of base editors to decipher drug mechanism of action. PLoS ONE 16(9): e0257537. https://doi.org/10.1371/journal.pone.0257537

Editor: Hodaka Fujii, Hirosaki University Graduate School of Medicine, JAPAN

Received: December 8, 2020; Accepted: September 5, 2021; Published: September 21, 2021.

Copyright: © 2021 Aparicio-Prat et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Sequencing data are available at Sequence Read Archive accession number SUB7836316 under BioProject ID PRJNA649296. http://www.ncbi.nlm.nih.gov/bioproject/649296.

Funding: The study was funded by Pfizer, Inc. The funder provided support in the form of salaries for authors EAP, DY, JPF, AW, HSX and RS, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

Competing interests: The authors EAP, DY, JPF, AW, HSX and RS were Pfizer employees. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Abbreviations: B2AR, β2 Adrenergic Receptor; Cas9, CRISPR Associated Protein 9; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; DNA, Deoxyribonucleic Acid; GFP, Green Fluorescent Protein; GPCR, G-protein coupled receptor; MoA, Mechanism of Action; MOI, Multiplicity of Infection; sgRNA, single guide Ribonucleic Acid.

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