FACS-based genome-wide CRISPR screening platform identifies modulators of CD47

Background: CD47 is a key innate immune checkpoint that enables tumor cells to evade macrophage-mediated clearance. Methods/Results: To systematically identify genetic regulators of CD47 surface expression, we performed FACS-based genome-wide CRISPR screens in three murine cancer cell lines B16 (melanoma), MC38 (colon adenocarcinoma), and EMT6 (breast carcinoma). Results: Comparative analysis of cells with high or low CD47 surface expression using DrugZ revealed CD47 itself as the top hit, validating the screens. Notably, DNAJC13 emerged as a consistent and robust regulator of CD47 expression across all three cell lines. Functional validation using DNAJC13-knockout cells confirmed a significant reduction in CD47 surface levels. Furthermore, in co-culture assays with macrophages, DNAJC13-deficient tumor cells exhibited increased susceptibility to phagocytosis, supporting a functional role for DNAJC13 in innate immune evasion. Finally, we verify that DNAJC13-knockout decrease tumor burden when treated with CD47 blockade. Conclusions: Overall, this study highlights a previously unrecognized regulator of CD47 and demonstrates the utility of high-throughput FACS-based CRISPR screening to uncover modulators of key immune checkpoint pathways.