PI: Gregory Hannon, Hannon Lab
Institution: Cold Spring Harbor Laboratory
Title: “An optogenetic toolkit for the interrogation and control of single cells.”
BRAIN Category: Tools for Cells and Circuits (RFA MH-14-216)
Dr. Hannon’s group will develop optogenetic techniques that use pulses of light to control genes and isolate proteins in specific cell types in the brain for molecular studies.
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.
Public Health Relevance Statement
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.
NIH Spending Category
Bioengineering; Biotechnology; Genetics; Mental Health; Neurosciences
Affect; Affinity; Affinity Chromatography; Animals; Antibodies; base; Behavior; Binding (Molecular Function); Biological; Biology; Biotin; Brain; brain cell; cell behavior; Cell Nucleus; cell type; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cultured Cells; Development; Dimerization; DNA; DNA Binding Domain; DNA-Binding Proteins; Drug Delivery Systems; Elements; env Gene Products; Epigenetic Process; Epitopes; Gene Expression; Gene Expression Alteration; Gene Transfer Techniques; Genetic; Genetic Materials; Genetic Transcription; Genome; Genomics; Goals; Grant; Human; In Vitro; in vivo; Infection; innovation; insight; Instruction; Ion Channel; irradiation; Label; Ligands; Light; Lipids; Messenger RNA; Methods; Modification; Molecular Genetics; multi-photon; mutant; Nature; Neurons; Neurosciences; new technology; nuclease; Nucleic Acid Binding; Nucleic Acids; optogenetics; Photons; Physiology; Process; Property; Proteins; public health relevance; Reading; receptor; reconstitution; Recovery; Recruitment Activity; Regulation; Resolution; Ribosomal Proteins; RNA; RNA Binding; S-nitro-N-acetylpenicillamine; Sampling; Site; Slice; Specificity; Specimen; Surface; Synapses; System; Technology; Tetracyclines; Tissues; tool; transcription factor; Transcriptional Regulation; Transgenes; Transgenic Mice; Translating; two-photon; Viral; Virus; Virus Diseases; Writing