The ability to form and maintain associations between environmental cues, actions, and rewarding stimuli is an elementary yet fundamental aspect of learned behavior that is necessary for survival. Multiple lines of research have identified that such reward-related learning is mediated by dopamine neurons located in the midbrain. Dopamine neurons encode stimulus-reward relationships in a way that is dynamically modified during learning, and impaired dopamine neuron functioning inhibits reward learning. However, almost nothing is known about how the molecular mechanisms in dopamine neurons that are relevant for learning. While pioneering studies have identified that Egr1 (Early growth response 1; an activity-responsive transcription factor) is rapidly upregulated in dopamine neurons after learning and correlates with learning strength, the functional consequence of this upregulation for learning and mechanistic consequences of this upregulation has not been examined. Specific Aim 1 of this proposal will use bidirectional CRISPR-based manipulation of Egr1 to determine it’s role in reward learning. Specific Aim 2 will define downstream gene targets of Egr1 induction using innovative single-cell sequencing as well as chromatin immunoprecipitation. This novel approach will demonstrate the necessity of Egr1 for learning and will pave the way for future experiments to explore how experience-dependent gene expression programs contribute to motivated behavior.