Our understanding of brain function at the cellular and circuit level is critically dependent on the ability to interrogate and alter neural cells withhigh specificity. The use of light, either through single-photon or multi- photon excitation, is the onl method that provides sufficient resolution to probe the brain at the cellular and subcellular levels. While light-activated molecules, like optogenetic proteins or photocaged compounds, have allowed many key insights in neuroscience, their use is still limited to those processes that can be affected by membrane channels. We propose to develop a toolkit allowing the interrogation, regulation, and modification of genetic information in brain cells using light. We wll build on a technology we have recently developed, LaserTag, based on the light-dependent interaction between protein tags (i.e. SNAP-tag or HALO-tag) and caged chemical ligands. Such interaction can be either use to recover molecules through affinity purification or to force the dimerization of proteins within a cell. Fusing SNAP and HALO to different cellular components will allow us to 1) Recover DNA and RNA from single cells for downstream analysis; 2) regulate transcription by recruiting activator and repressor domains to specific genomic loci; 3) deliver transgenes through viral infection with single cell resolution. The overall of these studies will b a broad new technology with the potential of transforming our ability to target specific cell types in the brain for genetic and molecular studies.
The ability to read, modify, and write genetic information at single-cell resolution would be transformative to neuroscience. We propose to develop a broad optogenetic toolkit, based on covalent protein tags and photoreleasable compounds, enabling the recovery of genetic material, the alteration of gene expression, and the insertion of transgenes to any cell of the brain with high spatial precision.
