The authors describe the development of a new adeno-associated virus (AAV) variant, rAAV2-retro, which enables efficient retrograde access to projection neurons in the brain. This variant was engineered through in vivo directed evolution to enhance its ability to transport viral particles retrogradely, i.e., from the periphery to the cell body of neurons. The rAAV2-retro variant shows up to two orders of magnitude improvement in retrograde transport compared to other AAV serotypes, comparable to the efficacy of synthetic retrograde tracers. The study demonstrates that rAAV2-retro can be used for functional interrogation of neural circuits, such as monitoring neural activity with calcium sensors or delivering gene editing tools for targeted genome modifications. The authors also explore the potential of rAAV2-retro for therapeutic applications, particularly in neurodegenerative diseases characterized by the spread of pathology through large-scale functional networks. Overall, the rAAV2-retro variant represents a significant advancement in the field of neural circuit dissection and gene therapy.The authors describe the development of a new adeno-associated virus (AAV) variant, rAAV2-retro, which enables efficient retrograde access to projection neurons in the brain. This variant was engineered through in vivo directed evolution to enhance its ability to transport viral particles retrogradely, i.e., from the periphery to the cell body of neurons. The rAAV2-retro variant shows up to two orders of magnitude improvement in retrograde transport compared to other AAV serotypes, comparable to the efficacy of synthetic retrograde tracers. The study demonstrates that rAAV2-retro can be used for functional interrogation of neural circuits, such as monitoring neural activity with calcium sensors or delivering gene editing tools for targeted genome modifications. The authors also explore the potential of rAAV2-retro for therapeutic applications, particularly in neurodegenerative diseases characterized by the spread of pathology through large-scale functional networks. Overall, the rAAV2-retro variant represents a significant advancement in the field of neural circuit dissection and gene therapy.