SUMMARY Cocaine addiction is driven by drug-induced adaptations in the function of mesolimbocortical brain reward circuits. A major unanswered question in the field of addiction biology is why behaviors such as drug seeking remain persistent even after cocaine use has ceased. A leading mechanistic hypothesis proposes that drug memory is encoded in the epigenome, and that consequent changes in gene expression within specific cell populations play a key role in the persistent circuit plasticities that drive addiction-associated behaviors. However, establishing experimental causality between the epigenome and drug seeking behaviors is challenging. In particular, the cellular heterogeneity of the brain presents a barrier to biochemical sequencing- based methods for chromatin studies, and the descriptive nature of epigenome data on its own is insufficient to demonstrate causality. We have been working to overcome these barriers via the application of innovative molecular genetic methods that allow us to both discover and disrupt chromatin regulation in specific populations of neurons in vivo. This study focuses on dopamine (DA) neurons of the ventral tegmental area (VTA), which play an essential role in the maintenance of drug seeking behaviors after cocaine exposure and forced abstinence. Transcription of the gene encoding Brain-Derived Neurotrophic Factor (Bdnf) is persistently induced in neurons of the VTA following forced abstinence after chronic cocaine, and the experimental elevation of VTA BDNF was shown to be sufficient to promote drug seeking in a rodent model of cocaine craving. Based on these data, we hypothesize that forced abstinence from cocaine induces functional changes in chromatin of VTA DA neurons that mediate the enhanced expression of plasticity genes including Bdnf. We further hypothesize that the enhanced transcription of Bdnf in the VTA is required for the induction of drug seeking. To test these hypotheses we will use an innovative low-input protocol to define the chromatin architecture of DA neurons from the VTA and to discover the changes in 3D chromatin architecture genome-wide that accompany persistent alterations in gene expression upon forced abstinence from chronic cocaine. Then we will use dCas9/CRISPR- mediated functional genome engineering to test the requirement for VTA Bdnf transcription in the maintenance of cocaine seeking behavior in a mouse model of the incubation of cocaine craving after forced abstinence. Taken together, these studies will use leading edge molecular genetic methods to advance circuit-level understanding of the cellular plasticities that contribute to increased drug seeking behaviors.