ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

PLoS Biol. 2020 Apr 10;18(4):e3000665. doi: 10.1371/journal.pbio.3000665. eCollection 2020 Apr.

Abstract

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Cells, Cultured
  • Dependovirus / genetics
  • Epitopes / genetics*
  • Female
  • Gene Editing / methods*
  • Gene Knock-In Techniques
  • Genes, Reporter
  • Genetic Vectors
  • Genome
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Male
  • Mice, Transgenic
  • Microscopy, Fluorescence
  • Molecular Imaging / methods
  • Neurons / immunology*
  • Neurons / physiology
  • Organ Culture Techniques
  • Proteins / genetics*
  • Proteins / immunology
  • Proteins / metabolism
  • Rats, Wistar
  • Receptors, N-Methyl-D-Aspartate / genetics
  • Receptors, N-Methyl-D-Aspartate / metabolism
  • Spatio-Temporal Analysis

Substances

  • Epitopes
  • Proteins
  • Receptors, N-Methyl-D-Aspartate
  • Green Fluorescent Proteins

Grants and funding

This work was supported by the Netherlands Organization for Scientific Research (ALWOP.191 to HDM and Graduate Program of Quantitative Biology and Computational Life Sciences to NS; https://www.nwo.nl/), the European Research Council (ERC-StG 716011; https://erc.europa.eu/funding/starting-grants), and the Brain and Behavior Research Foundation (NARSAD Young Investigator Award 24995; https://www.bbrfoundation.org/grants-prizes/narsad-young-investigator-grants) to HDM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.