Homo sapiens Raw sequence reads

The limited success of current immune-checkpoint blockade therapies emphasizes the need for new targets and combination treatments. VISTA is a promising immune checkpoint for cancer immunotherapy, but its regulation is poorly understood. To identify the key regulator for VISTA protein expression, we conducted an in vitro screen using a CRISPR KO library targeting ubiquitination-related proteins. HEK293T cells stably expressing VISTA-GFP fusion protein were efficiently transduced with the sgRNA library and sorted for GFP expression. Through in vitro human CRISPR KO screens targeting ubiquitination-related proteins and IP-coupled MS proteomic assays, our research successfully reveals TRIM25's role as the E3 ligase responsible for orchestrating VISTA protein stability.