Affiliations: [a] Brandenburg University of Technology Cottbus – Senftenberg, Institute of Biotechnology, Laboratory of Biochemistry, Senftenberg, Germany
| [b] Brandenburg University of Technology Cottbus – Senftenberg, Institute of Biotechnology, Laboratory of Molecular Biology, Senftenberg, Germany
Note:  These authors contributed equally to this work.
Abstract: The multifunctional proteasome activator PA28γ is involved in proteolytic pathways. Not surprisingly, upregulation of PA28γ contributes to tumor growth and progression. Therefore, CRISPR/Cas9 technology was used to specifically knockout the PSME3 gene encoding PA28γ in S462 tumor cells and HEK 293 cells. RNA guide sequences specific for PSME3 were designed and cloned into the lentiCRISPRv2 expression vector. Initially, successful Cas9-mediated gene-editing was verified by T7 endonuclease I DNA-mismatch assay. After the clonal selection, several clones showed no detectable PA28γ expression on protein level. Subsequently, a complete knockout of PA28γ was confirmed on genomic level by sequencing. Initial functional analysis of effector caspase activity in PA28γ knockout cells showed a higher basal activity and an increased sensitivity towards doxorubicin/ABT-737-induced apoptosis. Generally, this confirms the role of PA28γ as an anti-apoptotic regulator. To our knowledge, this is the first study addressing the role of proteasomal regulators in malignant peripheral nerve sheath tumor (MPNST) derived S462 cells.