Abstract 1372 The generation of an engineered HEK293T cell line bearing mutant NEMO incapable of binding to secondary binding site

NF-κB essential modulator (NEMO) is a necessary scaffold subunit of the Inhibitor of NF-κB Kinase (IKK) complex. IKK is central to the nuclear translocation of NF-κB, which is an inducible transcription factor that controls inflammatory gene expression. Activation of IKK catalytic activity in response to TNF-α and other canonical inducers of NF-κB requires formation of non-degradative linear polyubiquitin chains and their association with its NEMO subunit. Active IKK phosphorylates IκBα, an inhibitor protein of NF-κB, and triggers its degradation, thereby allowing NF-κB to enter the nucleus. Recent observations suggest that the IKK NEMO subunit, upon noncovalent association with linear polyubiquitin, mediates a second protein-protein interaction with the catalytic IKK2 subunit and "primes" the complex for activation. In order to investigate the direct involvement of NEMO in promoting catalytically active IKK, we have generated an engineered HEK293T cell line in which NEMO has been mutated to lack its ability to bind to this secondary protein-protein interaction. We used the CRISPR prime editing system to mutate the HEK293T cell line and verified the introduction of the mutation through Sanger sequencing and western blot. In future endeavors, the cell line will be used to compare to the normal wild-type cell line and the same engineering approach will be used to generate different mutations to monitor NEMO-mediated signaling events. Biochemistry Research at SDSU is supported in part by the California Metabolic Research Foundation.