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

Natalie Rimmer; Ching-Yeu Liang; Ricardo Coelho; Monica Nunez Lopez; Francis Jacob

doi:10.1016/j.xpro.2023.102305

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

STAR Protoc. 2023 May 12;4(2):102305. doi: 10.1016/j.xpro.2023.102305. Online ahead of print.

Authors

Natalie Rimmer¹, Ching-Yeu Liang², Ricardo Coelho², Monica Nunez Lopez², Francis Jacob³

Affiliations

¹ Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel 4031, Switzerland. Electronic address: natalie.rimmer@unibas.ch.
² Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel 4031, Switzerland.
³ Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel 4031, Switzerland; Hospital for Women, University Hospital Basel, Basel 4031, Switzerland. Electronic address: francis.jacob@unibas.ch.

Abstract

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.

Keywords: CRISPR; Cell Culture; Cell Separation/Fractionation; Flow Cytometry/Mass Cytometry; Gene Expression.