Bipolar disorder (BD) is a severe psychiatric disease characterized by recurrent mania, depression, and circadian rhythm disruption. Among circadian regulators implicated in mood-related dysfunction, Clock has emerged as a particularly strong mechanistic candidate. However, cell type-specific functions of Clock within mood-relevant circuits remain incompletely defined. Here, we developed and applied a Cre-dependent AAV-SaCas9 gene-editing strategy to disrupt Clock selectively in ventral tegmental area dopamine neurons. We first established a rapid in vitro screening pipeline for guide RNA selection that accurately predicted in vivo editing efficiency. We then targeted Clock in vivo using a single AAV-based editing strategy and observed robust titer-dependent reduction of Clock expression, by targeted sequencing, in situ hybridization, and immunohistochemistry. We assessed the functional consequences of Clock disruption across analysis levels, including a behavioral battery, circadian and sleep-wake measurements using EEG and EMG, and electrophysiological recordings. These results establish a practical framework for rapid, cell-type-specific disruption of candidate psychiatric risk genes and provide a mechanistically grounded model for investigating how loss of Clock function in mesolimbic dopaminergic circuits contributes to BD-relevant phenotypes.
Ju, Y. H., Zhao, J. Y., Sianati, S., Egebjerg, C., Gonzalez, O. C., Criswell, A., Lee, C. Y., Du, K., Luff, C. E., Stralka, J., Knapp, V., de Lecea, L., Kozorovitskiy, Y., Kauer, J. A., Fenno, L. E.
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