title: High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by In Vivo Genome Editing.
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.
authors.affiliation: Neuronal Signal Transduction Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA.
authors.forename: Takayasu
authors.initials: T
authors.lastname: Mikuni
authors.affiliation: Neuronal Signal Transduction Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA. Electronic address: jun.nishiyama@mpfi.org.
authors.forename: Jun
authors.initials: J
authors.lastname: Nishiyama
authors.affiliation: 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.
authors.forename: Ye
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Electron Microscopy Core Facility, Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA.
authors.forename: Naomi
authors.initials: N
authors.lastname: Kamasawa
authors.affiliation: Neuronal Signal Transduction Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA. Electronic address: ryohei.yasuda@mpfi.org.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Yasuda
mesh_terms: D001921:Brain / Q000196:embryology; D001923:Brain Chemistry; D001931:Brain Mapping / Q000379:methods*; D064113:CRISPR-Cas Systems; D005818:Genetic Engineering; D009419:Nerve Tissue Proteins / Q000032:analysis*; D059906:Neuroimaging / Q000379:methods; D009474:Neurons / Q000737:chemistry; D059010:Single-Cell Analysis
chemical_list: D009419:Nerve Tissue Proteins