Systematic experimental access to diverse neuronal cell types is a prerequisite to deciphering brain circuit organization, function, and dysfunction. Thus fundamental progress in neuroscience urgently needs cell type access technologies that are specific, comprehensive, easy to use, affordable, scalable, and general across animal species. Most if not all current genetic approaches to cell type targeting are based on genome and DNA engineering, which has inherent limitations in achieving the desired tool features. We have developed a paradigm-shifting technology for cell type targeting and manipulation based on RNA engineering. This technology builds upon the universal RNA sensing and editing system within metazoan cells, centered around the enzyme adenosine deaminase acting on RNA (ADAR). We term this method CellREADR: Cell access through RNA sensing by Endogenous ADAR. CellREADR can be deployed as a single RNA molecule that detects specific cellular RNAs through Watson-Crick base pairing and switches on the translation of markers, sensors, and effectors through a single base editing event; these RNA molecules can be delivered to animals via viral expression vectors. As such, CellREADR is highly specific and comprehensive, fast, cheap, easy to use, scalable, and in principle should apply to all animals. Importantly, CellREADR is inherently programmable, with unprecedented versatility for combinatorial and multiplexed targeting and editing of cell types in complex tissues. In this proposal, we will apply CellREADR to target and validate a large set of neuron types of the broadly defined cerebral cortex and basal ganglia in several mammalian and avian species. Our proposal is grounded on the evolutionary conservation as well as divergence of these forebrain cell types, which may underlie conserved and divergent circuit function and behavior across species. We have assembled an interdisciplinary team of investigators with expertise in molecular genetics, systems neuroscience, human and clinical neuroscience, bioengineering, and computation genomics. First, we will further optimize the CellREADR method and develop a comprehensive set of AAV tools for targeting and manipulating all major transcriptomic types of glutamatergic (GLU) and GABAergic neurons of the mouse cerebral cortex. Second, we will extend CellREADR to target and validate a large set of GLU and GABA neuron types in human ex vivo cortical tissues, macaque monkey cerebral cortex, and zebra finch cortex and basal ganglia. Third, we will establish a central CellREADR Portal for computational design of CellREADR reagents across vertebrate species and dissemination of technology and resource throughout the neuroscience community. By using cell-specific RNA profiles as the basis for genetic access and manipulation, CellREADR cell-editing technology is poised to transform the scale and rate of discovery in neuroscience and across biomedical fields. Impacts on the BRAIN Initiative will be immedi...