Optimized genetic tracers for viral mediated neuronal projection mapping.

Mapping fine axonal projections with high sensitivity remains a major challenge in neuroanatomy. Here, we developed Codon Optimized Membrane Embedded Tracers (COMET), a family of genetically encoded, membrane-anchored fluorescent proteins optimized for adeno-associated virus (AAV) delivery. COMET tracers—based on codon-optimized SuperFolder GFP (gCOMET) and TdTomato (rCOMET) fused to an H-RAS CaaX prenylation motif—enable robust plasma membrane targeting and enhance fluorescent signal localization to fine-caliber axons. In vivo, COMET tracers significantly outperformed conventional soluble fluorophores and chemical tracers in detecting corticospinal tract (CST) projections, revealing previously underappreciated collateralization and fine arborizations. Following spinal cord injury, COMET provided superior sensitivity for detecting regenerating and sprouting axons compared with biotinylated dextran amine (BDA). Importantly, COMET tracers retained bright native fluorescence after tissue clearing by CLARITY, enabling volumetric imaging of long-range projections without antibody amplification. COMET expands the available toolkit for high-resolution connectomics and regeneration studies, offering a versatile platform for sensitive, scalable, and cell-type-specific neuronal tracing in intact and injured mammalian nervous systems.