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Cell. 2016 Jun 16;165(7):1803-1817. doi: 10.1016/j.cell.2016.04.044. Epub 2016 May 12.

High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by In Vivo Genome Editing.

Author information

1
Neuronal Signal Transduction Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA.
2
Neuronal Signal Transduction Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA. Electronic address: jun.nishiyama@mpfi.org.
3
Neuronal Signal Transduction Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA; Integrative Program in Biology and Neuroscience, Florida Atlantic University, Jupiter, FL 33458, USA.
4
Electron Microscopy Core Facility, Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA.
5
Neuronal Signal Transduction Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA. Electronic address: ryohei.yasuda@mpfi.org.

Abstract

A scalable and high-throughput method to identify precise subcellular localization of endogenous proteins is essential for integrative understanding of a cell at the molecular level. Here, we developed a simple and generalizable technique to image endogenous proteins with high specificity, resolution, and contrast in single cells in mammalian brain tissue. The technique, single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (SLENDR), uses in vivo genome editing to insert a sequence encoding an epitope tag or a fluorescent protein to a gene of interest by CRISPR-Cas9-mediated homology-directed repair (HDR). Single-cell, HDR-mediated genome editing was achieved by delivering the editing machinery to dividing neuronal progenitors through in utero electroporation. We demonstrate that SLENDR allows rapid determination of the localization and dynamics of many endogenous proteins in various cell types, regions, and ages in the brain. Thus, SLENDR provides a high-throughput platform to map the subcellular localization of endogenous proteins with the resolution of micro- to nanometers in the brain.

PMID:
27180908
PMCID:
PMC4912470
DOI:
10.1016/j.cell.2016.04.044
[Indexed for MEDLINE]
Free PMC Article
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