G protein-coupled receptors (GPCRs) are essential signal transducers within cells, responsible for relaying a wide variety of messages across the cell surface to influence cell behavior. Dysregulation of GPCRs is linked to developmental defects and numerous diseases. As a consequence, the behavior of GPCRs is tightly controlled by a network of interacting or modulatory proteins that help GPCRs localize and signal correctly. The advent of CRISPR represents an opportunity to use high-throughput, unbiased approaches to uncover novel modulators of GPCR signaling. The Hedgehog (Hh) signaling pathway is a GPCR-associated signaling pathway critical for vertebrate development and adult tissue homeostasis. Numerous questions remain about how the activity and localization of Smoothened (Smo)—a core Hh pathway component and GPCR—is controlled and modulated in response to Hh ligand. Here I present the results of a CRISPR screen of lipid-related genes that uncovered several lipid metabolic pathways that influence Hh signaling activity through separate specific effects on Smo. In chapter two, I present evidence that sphingomyelin synthesis and cholesterol synthesis both impact Hh signaling by modulating Smo activity, a discovery that also provides insight into a central question remaining about the mechanisms of vertebrate Hh signaling: how can cholesterol, an abundant lipid in the plasma membrane, act as the endogenous ligand for Smo as has been hypothesized? Using a combination of pharmacological and genetic approaches, we establish that inhibition of sphingolipid synthesis potentiates Hh signaling, and cholesterol biosynthesis inhibition attenuates signaling. Epistasis experiments indicate that these pathways impact Hh signaling at the level of Smo. Toxin-based fluorescent sensors of membrane lipids allowed us to dissect the mechanisms behind these observations. An increased sphingomyelin to cholesterol ratio in the ciliary membrane in comparison to the plasma membrane limits cholesterol accessibility in that organelle due to sphingomyelin’s ability to sequester cholesterol and reduce its chemical activity. Sphingolipid synthesis inhibition therefore potentiates Hh signaling by reducing this ratio and, as a result, increasing levels of accessible ciliary cholesterol. We postulate that this small pool of accessible cholesterol—rather than the total membrane cholesterol—is responsible for activating Smo and driving Hh signaling. v Hh ligand increases cholesterol accessibility and therefore Smo activity by inhibiting the activity of Patched1. Thus, a focused CRISPR screen revealed that two lipid biosynthetic pathways impact Hh signaling via a specific effect on Smo activity. Further, this work demonstrates that cholesterol organization within the ciliary membrane can have a profound impact on ciliary signaling. In chapter three, I validate another prospective negative regulator of Hh signaling suggested by our focused CRISPR screen: the N-glycan synthesis pathway. Most GPCRs are believed to be N-glycosylated, and this post-translational modification can have great functional importance. Despite these facts, the impact of N-glycosylation remains poorly understood for many proteins. I demonstrate that inhibition of this general pathway has a specific effect on Hh pathway activity, potentiating signaling by increasing Smo accumulation at the ciliary membrane in response to Hh ligand. The impact of N-glycan synthesis inhibition on Smo is not a result of hypoglycosylation of Smo itself, but rather the result of loss of glycosylation on an alternative protein. In addition, the effect of this perturbation is cilia-dependent. Thus, inhibiting this general lipid-associated process again has a significant and specific effect on Smo, in this case altering its localization during key parts of the signaling process. GPCR-mediated signaling is a conserved critical component of cellular function. In this work, I advance our understanding of how a particular GPCR-mediated signaling pathway—the Hh pathway—is regulated. In addition, our CRISPR screen sheds light on specific impacts of three lipid-related pathways on Hh signaling, suggesting that this methodology may be useful to interrogate the relationship between lipid metabolic networks and GPCR signaling more generally.