293T CRISPR/Cas9 genome wide screen to identify regulators of TNF shedding

TNF (Tumor Necrosis Factor) is a pro-inflammatory cytokine involved in the regulation of immune responses but also in the development of inflammatory diseases. TNF is produced as a membrane protein that needs to be cleaved by membrane metalloproteases, mainly ADAM17 (A Disintegrin and Metalloprotease 17), in order to form the mature cytokine. ADAM proteases cleave multiple substrates on the cell membrane, thereby contributing to many biological processes. For this reason, their activity is tightly regulated by the cells. Due to their role in physiological and pathological aspects, both TNF and ADAM proteases are matter of intense research. By using a genetically encoded reporter, C-Tag TNF , we performed a CRISPR/Cas9 genome wide screen in 293T cells for the identification of genetic factors involved in TNF shedding. The C-Tag TNF reporter is based on the presence of a cryptic epitope that can be detected by a nanobody only upon TNF cleavage by ADAM proteases. After transduction with a genome-wide library, 1% of cells showing the lowest C-Tag signal in the KO pool were sorted, followed by deep sequencing to identify gRNAs enriched in the "no shedding population" compared to the unsorted negative control.