AbstractCirculating tumor cells (CTCs) break free from primary tumors and travel through the bloodstream and lymphatic system to seed metastatic tumors, which are the major cause of death from cancer. The identification of the major genetic factors that enhance production and persistence of CTCs in the bloodstream at a whole genome level would enable more comprehensive molecular mechanisms of metastasis to be elucidated and the identification of novel therapeutic targets, but this remains a challenging task due to the heterogeneity and extreme rarity of CTCs. Here, we describe the firstin vivogenome-wide CRISPR KO screen using CTCs directly isolated from a mouse xenograft. This screen elucidatedSLIT2– a gene encoding a secreted protein acting as a cellular migration cue – as the most significantly represented gene knockout in the CTC population.SLIT2knockout cells are highly metastatic with hypermigratory and mesenchymal phenotype. Reduced expression ofSLIT2is observed in human tumors, indicating its role as a negative modulator of tumor progression and metastasis.