Creation of CD63-deficient HEK293 cell lines using a polycistronic CAS9/EGFP/HSVtk/PuroR expression vector

Abstract

CD63 is an integral membrane protein that is present at the plasma membrane but highly enriched in endosomes and lysosomes. CD63 has been implicated in several cell biological processes and is a common constituent of small, secreted vesicles (diameter of ~30-300 nm) known as exosomes. Here, we report a poly-cistronic Cas9/EGFP/HSVtk/PuroR expression vector that is designed to allow for (i) guide RNA (gRNA)-mediated mutation of a specific gene, (ii) puromycin selection of Cas9-expressing cells, (iii) Cre-mediated excision of the Cas9-encoding ORF, and (iv) ganciclovir killing of Cas9-expressing cells. It was further modified to express gRNAs that target the human CD63 gene. This plasmid was transfected into HEK293 cells followed by the expansion of single cell clones (SCCs). Immunoblot analysis revealed that multiple SCCs lacked detectable levels of CD63 expression. Four such CD63-deficient SCCs were chosen for further analysis. In each of three or more trials, these SCCs were found to lack CD63 protein expression. Furthermore, these four CD63-deficient cell lines all had DNA insertions or deletions in exon 2 of this gene that shifted the reading frame, precluded the expression more than the first 44-46 amino acids of CD63, and are predicted to induce nonsense-mediated decay of the CD63 mRNA.