Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining

Abstract

Summary: We provide a protocol using non-homologous end joining to integrate an oligonucleotide sequence of a fluorescence protein at the CDH1 locus encoding for the epithelial glycoprotein E-cadherin. We describe steps for implementing the CRISPR-Cas9-mediated knock-in procedure by transfecting a cancer cell line with a pool of plasmids. The EGFP-tagged cells are traced by fluorescence-activated cell sorting and validated on DNA and protein levels. The protocol is flexible and can be applied in principle to any protein expressed in a cell line.For complete details on the use and execution of this protocol, please refer to Cumin et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Culture
• Flow Cytometry/Mass Cytometry
• Cell Separation/Fractionation
• Gene Expression
• CRISPR