PROJECT SUMMARY Determining the subcellular localization of a protein under different cellular states is a critical aspect of assessing protein function and dysfunction. This is especially important in neuroscience as neurons are complex, polarized cells with distinct functional compartments, including synapses. Synapse dysfunction underlies many neural and psychiatric disorders. Interestingly, synapses connecting different neurons develop unique structural and functional properties that differentially modulate circuit function. This structural and functional diversity is mediated by molecular differences. However, our understanding of the proteins located at different types of synapses is very limited. Suitable antibodies are simply not available for many proteins and protein overexpression drives mis-localization. Therefore, methods to localize endogenous synaptic proteins in brain tissue are urgently needed. Here, we developed a CRISPR gene editing strategy that, in one seamless genetic modification, inserts an epitope tag onto a protein of interest and drives expression of a cell marker in postmitotic neurons using a single AAV. Our innovative new method is the first to provide an integrated means for selectively identifying only those neurons that correctly integrated the protein tag and provide a cell filling, structural reference necessary for determining the synapse-specific localization of a protein. Our method is highly flexible for a variety of proteins, tags, and cell markers. Preliminary data indicate that our method correctly tags the synaptic protein N-cadherin in cultured neurons but it requires further optimization and expansion in vitro (Aim 1) and in vivo (Aim 2). Successful completion of our proposal will yield new technologies that allow the study of endogenously expressed, synapse-specific proteins in the brain. As an example, we will test the hypothesis that different cadherins associated with distinct mental illnesses localize to different types of synapses in vivo. Taken together, our results are expected to result in a new technology that can be broadly applied to study synapse formation and function and provide new molecular insight to mechanisms underlying synapse diversity.