Ribozyme-optimized CVS-N2cΔG Rabies Virus with Hyper-diverse Barcodes for High-fidelity Single‑Neuron Projection Connectivity

Abstract Neural circuit mapping at single‑cell resolution relies on hyper‑diverse barcode libraries to minimize collision artifacts and topological distortion. Here, we report a minigenome‑based screening platform for systematic optimization of ribozyme configurations in the CVS‑N2c rabies virus strain. Screening of combinatorial ribozyme libraries identified 3′ Twister‑based configurations that increased CVS‑N2cΔG rescue efficiency by two orders of magnitude and expanded effective library capacity by 75‑fold relative to conventional systems. We applied these optimized hyper‑diverse barcode libraries to the mouse LGN input to V1 visual pathway and assessed both library composition and downstream biological connectivity using collision‑free, cell‑centric network topology analysis. Twister‑engineered hyper‑diverse libraries suppressed collision‑induced false‑positive spurious convergence, recovering a network architecture consistent with the sparse, parallel organization of LGN input to V1 corticothalamic feedback. Together, these results demonstrate that ribozyme‑mediated barcode diversity enhancement and collision‑free topology reconstruction act synergistically: higher library diversity maximizes the absolute yield of collision‑free cells, while cell‑centric filtering resolves residual false‑positive hubs, together enabling high‑fidelity single‑neuron projection connectivity at scale.