A designer AAV variant, rAAV2-retro, enables efficient retrograde access to projection neurons, offering a powerful tool for neural circuit analysis and gene therapy. This variant was developed through in vivo directed evolution to enhance retrograde transport capabilities of adeno-associated virus (AAV) capsids. The rAAV2-retro variant efficiently delivers sensors and effectors to projection neurons, enabling functional circuit interrogation and in vivo genome editing. It matches the efficacy of classical retrograde tracers and supports high-level transgene expression in targeted neuronal populations. The study demonstrates that rAAV2-retro provides robust retrograde access to projection neurons in various circuits, including the corticopontine tract, with efficiency comparable to synthetic retrograde tracers like Fluoro-Gold. It also shows broad applicability across different brain regions and circuits, including the dorsomedial striatum. The variant can be combined with Cre recombinase driver lines to enable precise targeting of specific neuronal populations, enhancing the specificity of circuit interrogation. rAAV2-retro is also effective for monitoring neural activity using genetically encoded indicators like GCaMP6f and for gene manipulation through CRISPR/Cas9 systems. It allows for efficient gene editing in projection neurons, demonstrating its potential for therapeutic applications. The variant's ability to access a wide range of projection neurons makes it a valuable tool for understanding neural circuit function and for future therapeutic interventions in neurodegenerative diseases. The study highlights the potential of rAAV2-retro as a versatile and efficient tool for both basic research and clinical applications in neuroscience.A designer AAV variant, rAAV2-retro, enables efficient retrograde access to projection neurons, offering a powerful tool for neural circuit analysis and gene therapy. This variant was developed through in vivo directed evolution to enhance retrograde transport capabilities of adeno-associated virus (AAV) capsids. The rAAV2-retro variant efficiently delivers sensors and effectors to projection neurons, enabling functional circuit interrogation and in vivo genome editing. It matches the efficacy of classical retrograde tracers and supports high-level transgene expression in targeted neuronal populations. The study demonstrates that rAAV2-retro provides robust retrograde access to projection neurons in various circuits, including the corticopontine tract, with efficiency comparable to synthetic retrograde tracers like Fluoro-Gold. It also shows broad applicability across different brain regions and circuits, including the dorsomedial striatum. The variant can be combined with Cre recombinase driver lines to enable precise targeting of specific neuronal populations, enhancing the specificity of circuit interrogation. rAAV2-retro is also effective for monitoring neural activity using genetically encoded indicators like GCaMP6f and for gene manipulation through CRISPR/Cas9 systems. It allows for efficient gene editing in projection neurons, demonstrating its potential for therapeutic applications. The variant's ability to access a wide range of projection neurons makes it a valuable tool for understanding neural circuit function and for future therapeutic interventions in neurodegenerative diseases. The study highlights the potential of rAAV2-retro as a versatile and efficient tool for both basic research and clinical applications in neuroscience.