We propose to develop a synthetic biological system to manipulate subcellular RNAs and proteins in growth cones or synapses of subtype- and context-specific neurons. This system has both direct, powerful near-term experimental-investigative potential and future potential toward novel, uniquely specific forms of therapeutics/ bioactive delivery. The systems enable unique forms of subcellular functional investigation in subtype- and stage- specific neuronal circuitry, offering generalizable modularity for manipulation of RNA and/or protein localized to developing growth cones during circuit formation and to presynaptic compartments in maturing and mature circuitry. Subcellular functions of proteins regulating connectivity and (dys)function of subtype-specific circuitry are poorly understood. Based on successful preliminary studies, we propose to develop these tools for in vivo perturbation of molecular abundances by integrating subcellular localization motifs with inducible gene expression systems. This system will enable discovery of RNAs/proteins that function subcellularly to control overall organization, precision, and function of distinct neural circuits, and how they are dysregulated in disease. We aim to integrate motifs that selectively traffick proteins to GCs and/or presynapses with an inducible gene expression system, enabling subcellular-specific overexpression via direct motif fusion, and knockdown via motif fusion to hfCas13d, a refined mRNA degrader. We will use data from recently developed experimental and analytical approaches purifying subtype-specific GCs or presynapses and their parent somata or nuclei from developing or mature mouse cortex, and quantitatively “mapping” RNAs and proteins between these subcellular compartments. This enabled identification of molecules with subcellular localizations specific to subtypes and stages, and/or to disease, and measuring of local translation. However, such candidates can function in multiple subcellular domains, at multiple distinct stages/contexts. Current approaches for whole-neuron perturbation of molecular abundances produce off-target effects, complicating investigation of subcellular-specific RNA/protein function, thus preventing elucidation of molecular mechanisms regulating circuit formation and (dys)function. We aim to develop approaches for subcellular- and stage-/context-specific manipulation of molecular abundances in developing and mature circuits. In Aim 1, we will develop a system to subcellularly manipulate RNA/protein abundances in growth cones that regulate distinct developmental stages of circuit construction. In Aim 2, we will develop a system to subcellularly manipulate presynaptic RNA/protein abundances later in maturity that regulate synapse function, maintenance, and plasticity, thus are linked to memory, synaptic weighting, and circuit function. We will employ presynaptic-localizing motifs in mature circuits. These systems will enable elucidation of subcellular...