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 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.