One-step approach producing barcoded rabies virus with optimized diversity

Mapping brain-wide neuronal connectivity is essential for understanding brain function, and barcoded rabies virus offers a powerful tool for this purpose. However, their application has been hindered by challenges in achieving sufficient barcode diversity and efficient transsynaptic transfer. While the CVS-N2cΔG strain offers improved transsynaptic transfer capabilities, producing barcoded versions of this strain has remained technically demanding. Here, we introduce an alternative one-step method for producing SAD-B19ΔG and CVS-N2cΔG strains. This streamlined approach simplifies the production process, significantly reduces production time, and eliminates background contamination. It improves the diversity and uniformity of the rabies virus barcode library. Moreover, the tracing efficiency of viruses produced by this one-step method matches that of conventional techniques. By addressing these limitations, our approach benefits the future development and application of barcoded-rabies-virus-based connectomic studies.