Disruption of ligand binding domain of the Epidermal Growth Factor Receptor by sequential knock in CRISPR/Cas9 genome editing alters its subcellular localization and phosphorylation in cervical cancer cells
Upregulation of Epidermal Growth Factor Receptor is evident in most cases of cervical cancer and is usually associated with a poor prognosis. At the same time, therapies directed against EGFR are generally not successful in this type of cancer, which suggests that therapeutic inactivation of EGFR can be easily overcome. In order to evaluate mechanisms by which cervical cancers with alterations in EGFR signaling thrive, we have generated several EGFR mutant clones by CRISPR/Cas9 genome editing. This work details the approach used to generate EGFR mutant cells and describes changes in cell characteristics associated with decreased quantity and altered subcellular distribution of EGFR. Overall design: We performed CRISPR/Cas9 experiments with the intention to introduce amino acid substitutions into EGF binding region of EGFR gene in cervical cancer cell line ME180 genome. We porformed Whole genome sequencing in order to get full insight into CRISPR/Cas9 results in 5 single cell isolated clones  The aim of this approach was to precisely evaluate the variants of EGFR across the entire EGFR gene region and to compare the entire genomes between the clones and wild type cells.