Peer reviewed: TrueAcknowledgements: We thank Elena Williams for comments on the manuscript. We thank Jerome Boulanger for writing the script for the 3d-alignment of 2-photon recordings, Nicolas Alexandre for the help with the bioinformatic analysis of the NGS datasets, the Laboratory of Molecular Biology (LMB) workshops for the help with software and hardware development, and members of the Biological Service Group for their support with the in vivo work. This study was supported by the Medical Research Council (MC_UP_1201/2), the European Research Council (STG 677029 to MT), the European Union’s Horizon 2020 research and innovation program with the Marie Sklodowska-Curie fellowship to DdM (894697), the Cambridge Philosophical Society and St. Edmund’s College (University of Cambridge) with the Henslow Research Fellowship to AGR, the Rosetrees Trust with an MBPhD fellowship to HL (M598). For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising. All data are stored on the LMB server. All materials described in this paper can be obtained upon reasonable request and for non-commercial purposes after signing a material transfer agreement (MTA) with the MRC.Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that "revertant" mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments
