Supplement for Center for Synthetic Regulatory Genomics: Building CACNA1C alleles associated with Neuropsychiatric Disorders

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Project Summary The Center for Synthetic Regulatory Genomics (SyRGe) is tasked with development and application of technology for making coordinated changes to large gene loci, broadly enabling investigations into the function of regulatory sequences revealed by genome wide association study (GWAS) “hits”. The Center’s tools dramatically supersede present technologies for manipulation and assessment of regulatory genome function through its focus on assembly and delivery of so-called Big DNA. This supplement request proposes to leverage our most recent technology development in Big DNA assembly and delivery to tackle a large locus in CACNA1C, containing the top hits associated with various neuropsychiatric disorders, including schizophrenia, bipolar disorder, major depression, ADHD and ASD. CACNA1C encodes a pore-forming subunit of the L-type voltage-gated calcium channel and is widely expressed in neurons, endocrine cells, cardiomyocytes and smooth muscles. Calcium influx through the channel is critical for synaptic plasticity, gene expression regulation, insulin secretion and muscle contraction. In this supplement request, we propose to assemble and deliver the ~330 kb human CACNA1C intron 3 to replace the mouse endogenous Cacna1c intron 3. We will build and deliver human CACNA1C intron 3 that contains risk or protective alleles and differentiate the engineered mouse ESCs into neurons for initial phenotyping. We will also build a series of variants for further functional studies. We will assess the Cacna1c mRNA and L-type calcium current in differentiated neurons. The proposed work in this supplement request will enable insights into the molecular mechanisms underlying multiple neuropsychiatric disorders, and it will also serve as the foundation for further generation of humanized Cacna1c mouse models for behavioral studies.