Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain.

Elevated Epidermal Growth Factor Receptor (EGFR) expression is observed in most cervical cancers, and it is frequently associated with poor clinical outcomes. The limited efficacy of existing EGFR-targeted therapies in cervical cancer highlights the need for a deeper understanding of EGFR role in this cancer type. To investigate EGFR separately from its interaction with Epidermal Growth Factor (EGF), we removed the key amino acids from the ligand bindings site. We used CRISPR/Cas9 genome editing to generate a panel of EGFR mutant cell lines and then sequenced and characterized them in detail. Studying the phenotypes of mutant cell clones, we show that a pair of amino acid substitutions L14R and Y45M within Domain I of EGFR protein completely disrupts EGF binding and changes EGFR subcellular distribution. A single substitution Y45M significantly reduced EGF binding but did not lead to subcellular redistribution of EGFR. Upon editing, EGFR mRNA and protein expression were decreased in mutant clones compared to wild type cells. Genome wide profiling of different CRISPR/Cas9 clones confirmed correct editing of EGFR with no off target CRISPR/Cas9 generated mutations. At the same time, spontaneous mutations that could impact cell phenotypes were detected in mutant clones. Disruption of ligand binding domain of EGFR by sequential knock in CRISPR/Cas9 genome editing altered subcellular localization and phosphorylation of EGFR in cervical cancer cells. The results presented here provide insights that may accelerate the development of CRISPR/Cas9-based therapies for EGFR-dependent cancers and reinforce the importance of thorough evaluation of CRISPR/Cas9-generated phenotypes.