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Cis-regulatory elements such as enhancers play critical regulatory roles in modulating gene expression during brain development. The development of massively parallel reporter assays has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted the self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identify putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. In vivo functional testing of putative enhancers, as we demonstrate here, will be critical toward characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in normal and pathogenic brain development.

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