CRISPR Screens Uncover Genes that Regulate Target Cell Sensitivity to the Morphogen Sonic Hedgehog

To uncover regulatory mechanisms in Hedgehog (Hh) signaling, we conducted genome-wide screens to identify positive and negative pathway components and validated top hits using multiple signaling and differentiation assays in two different cell types. Most positive regulators identified in our screens, including Rab34, Pdcl, and Tubd1, were involved in ciliary functions, confirming the central role for primary cilia in Hh signaling. Negative regulators identified included Megf8, Mgrn1, and an unannotated gene encoding a tetraspan protein we named Atthog. The function of these negative regulators converged on Smoothened (SMO), an oncoprotein that transduces the Hh signal across the membrane. In the absence of Atthog, SMO was stabilized at the cell surface and concentrated in the ciliary membrane, boosting cell sensitivity to the ligand Sonic Hedgehog (SHH) and consequently altering SHH-guided neural cell-fate decisions. Thus, we uncovered genes that modify the interpretation of morphogen signals by regulating protein-trafficking events in target cells. [Display omitted] •CRISPR screens identify positive and negative regulators of Hedgehog signaling•Screens identified known and new genes linked to cilia and ciliopathies•Atthog, Megf8, and Mgrn1 attenuate target cell responses to Hedgehog signals•Attenuators of signaling promote the endocytosis and degradation of Smoothened Pusapati et al. use CRISPR-based screens in mammalian cells to identify genes that modify cellular responses to Hedgehog morphogens. The screens provide a comprehensive view of the regulatory structure of Hedgehog signaling, highlight its intimate connection to primary cilia and ciliopathies, and uncover a role for Smoothened trafficking in regulating signaling strength.