RNA Programmable and Scalable Brain Cell Type Tools Across Vertebrates

Duke University, Durham NC

Investigators

Abstract

ABSTRACT A major goal in neuroscience is to analyze neural circuit structure and function in diverse brain regions and species with cell type resolution. Achieving this goal requires novel technologies and reagent resources that grant cell type access yet are scalable and generalizable across brain regions and species. Transcriptional regulation of gene expression is a fundamental and universal mechanism underlying cell type specification across species and life spans. Most current approaches to gain access to cell types attempt to recapitulate RNA expression patterns through DNA engineering of transcriptional regulatory elements (e.g. enhancers). However, the generality and scalability of the enhancer approach, especially to other non-model vertebrate species, remain to be established. We recently invented an orthogonal RNA and translation-based method that represents a new paradigm in cell type technology. CellREADR/RADARS is built upon the universal RNA editing system within all metazoan cells mediated by the enzyme adenosine deaminase acting on RNA (ADAR). CellREADR is deployed as a single modular RNA molecule that detects a specific cellular RNA through RNA-RNA base pairing and then switches on the translation of effector proteins to monitor and manipulate the cell. Notably, CellREADR can be delivered efficiently to brain tissues via viral vectors. As such, CellREADR is potentially highly specific, easy to use, exceptionally scalable and programmable, and applicable to all vertebrates. Fully realizing the enormous potential of CellREADR requires a scalable and generalizable platform for systematically screening and validating a large number of RNA sensors for all major cell types across brain regions and vertebrate species. Here we have assembled an exemplary team of molecular geneticists, systems and evolutionary neurobiologists, and a physicist to establish a spatial transcriptomics-based pipeline that is scalable and generalizable for high-throughput screening of RNA sensor libraries in the brains of different vertebrate taxa. We will first apply this pipeline to identify hundreds of cell type RNA sensors across marmoset, mouse, songbird, and salamander brain. We will focus on cell types of the cerebral cortex/pallium, basal ganglia, and thalamus, as these structures give rise to the cortico- basal ganglia-thalamic circuit loops that are highly conserved across vertebrates and fundamental to sensory, motor, cognitive, and emotional functions. We will then validate the specificity, efficiency, and functionality of these RNA sensors. Finally, we will register these sensor libraries with current cell type atlases in each species and catalog them with validation information in a format that facilitates dissemination through the BRAIN Armamentarium network and other publicly accessible portals. Our project is poised to transform vertebrate neuroscience through cell type and circuit resolution comparative analysis and will lay a path to cell type-targeted gene therapy and circuit modulation in neuropsychiatric diseases. 1