ABSTRACT Experimental access to specific cell classes or types in the brain is essential for elucidating their roles in neural circuit function. Single cell transcriptomics has enabled unprecedented definition of cell types at the molecular level and has uncovered numerous marker genes, which can be used to generate genetic tools for access to specific cell populations. However, high-precision genetic tools for experimental access to specific cell-types still rely on genome modification and subsequent crossing of animals harboring different transgenes to generate experimental animals. Specificity can also be achieved by stereotaxic injection of viruses, but this approach still usually requires a combination with transgenes, and produces only local infection which can result in significant expression variability between animals and can cause reactive gliosis. Animal crosses are laborious and inefficient, especially if more than two transgenes are needed to achieve specific cell-type labeling. We propose to develop a suite of enhancer viruses that can provide access to specific brain cell types or classes to the whole brain when delivered by retroorbital injections. The viruses will be endowed with specificity by containing enhancers defined experimentally from the combination of single-cell RNA-seq and single cell ATAC-seq data (Assay for Transposase-Accessible Chromatin with next generation sequencing). The viruses will be screened for specificity by several methods including whole-brain imaging, single-cell RNA-seq and multiplex FISH. Intersectional genetic approaches to further refine specificity and enable functional interrogation of specific cell types will also be established. DNA constructs, virus packaging techniques, recommended titers, and whole- brain expression data will be made publicly available.

PROJECT NARRATIVE Experimental access to specific cell classes or types in the brain is essential for elucidating their roles in neural circuit function. We propose to employ a newly developed approach in our lab to enable cell class- or type- specific access by enhancer-driven adeno associated viruses in a brain-wide fashion. These viruses have the potential to replace many equivalent transgenic lines in neural circuit studies, and to dramatically facilitate the access to specific cell types due to their ease of generation, sharing and use.