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

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project--Cooperative Agreements (U01)
Project #
1U01MH106035-01
Application #
8822629
Study Section
Special Emphasis Panel (ZMH1-ERB-L (04))
Program Officer
Freund, Michelle
Project Start
2014-09-26
Project End
2017-06-30
Budget Start
2014-09-26
Budget End
2015-06-30
Support Year
1
Fiscal Year
2014
Total Cost
$452,400
Indirect Cost
$202,400
Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724