Abstract:
Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR/Cas9, which was originally characterized as an adaptive immune system in bacteria, has become the most powerful genome engineering technology in recent years and it can be used as a genome engineering and knockout technology due to its ease of use and flexibility. CRISPR/Cas9 can also be turned into a powerful gene expression manipulation tool by conversion of Cas9 endonuclease into a catalytically inactive form called dead-Cas9 (dCas9). When dCas9 is fused with transcriptional repressor or activator domains, it is able to supress or activate the target gene expression, respectively with the design of proper target sites and guide RNAs (sgRNA). In this study, we aimed to set up CRISPR based knockout, inhibition (CRISPRi) and activation (CRISPRa) systems and implement them in melanoma cells to characterize the role of Interferon Regulatory Factor 4 (IRF4) in in vitro assays that interrogate cancerrelated phenotypes. In this study, we successfully set up lentiviral and inducible CRISPR based knockout, knockdown (CRISPRi) and activation (CRISPRa) methods, which were used to manipulate IRF4 expression in melanoma cell lines. After validation of these systems, we used them in GFP competition assays. According to our preliminary data, IRF4 is important for the competitive fitness and/or survival of melanoma cells, which is in line with a model where melanoma cells have non-oncogene addiction to IRF4 expression, similar to several B-cell origin cancers.
