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

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 polycistronic 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. Introduction Like all tetraspanins [1], CD63 is co-translationally translocated into the endoplasmic reticulum as a multipass, four transmembrane-domain protein with its Nand Cterminus oriented in the cytoplasm. CD63 is subsequently trafficked to the plasmamembrane, endosomes, multivesicular bodies (MVBs), and the lysosomes that they form, but is particularly enriched in endosomes, MVBs, and lysosomes [2]. CD63 has been implicated in a number of cell biological processes, including endo/lysosomal sorting of its partner proteins [3] and the biogenesis of multivesicular bodies [4]. CD63 is also a common constituent of small secreted vesicles known as exosomes [5]. To better understand the role(s) of CD63 in human cell biology, we created a Cas9-expressing plasmid designed to introduce mutations in the CD63 gene due to its simultaneous expression of CD63-directed guide RNAs [6] [7]. This Cas9/CRISPR vector co-expresses Cas9-3xNLS in a polycistronic ORF together with EGFP, Herpes Simplex Virus thymidine kinase, and the puromycin-resistance enzyme, each separated by viral 2a peptides. This plasmid is designed for the Cas9/gRNA-mediated mutation of the CD63 gene, but also for the positive selection of Cas9 ORF-expressing cells, Cre recombinase-mediated deletion of the Cas9 ORF, and the positive ganciclovir selection of cells that no longer express this ORF. This plasmid successfully used to induce mutations in the human CD63 gene in HEK293 cells, and we report the nucleotide sequences of 4 CD63-deficient HEK293 cell lines. Objective The objectives were to (1) generate a Cas9/gRNA expression plasmid that can be used to generate CD63-deficient human cells and (2) use this plasmid to generate CD63-deficient HEK293 cell lines. Creation of CD63-deficient HEK293 cell lines using a polycistronic CAS9/EGFP/HSVtk/PuroR expression vector DOI: 10.19185/matters.201604000004 Matters (ISSN: 2297-8240) | 2 a