Single-cell labeling of endogenous proteins in the mammalian brain by in vivo genome editing [with video]

A scalable and high-throughput method to determine the localization of endogenous proteins is essential to the understanding of a cell at the molecular level. A new method termed SLENDR, which is based on in vivo genome editing in the mammalian brain, has been developed to visualize the localization of endogenous proteins in single brain cells.

HFSP Long-Term Fellow Takayasu Mikuni and colleagues
authored on Thu, 02 June 2016

Thousands of proteins are known to mediate functional processes in a cell. Thus, precise, rapid and comprehensive mapping of subcellular localization of endogenous proteins is essential to describe cellular processes at the molecular level. However, conventional techniques have not provided rapid, scalable and high-throughput readouts for the localization of endogenous proteins, especially in the mammalian brain, due to the extremely high density and complexity of cellular processes.

Figure: Strategy and application of SLENDR.

Direct, single-cell modification of the genome in vivo to insert a tag sequence to a gene of interest would provide rapid, specific and sparse labeling of the gene product protein. Using this strategy, 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, termed SLENDR (single-cell labeling of endogenous proteins by CRISPR-Cas9-mediated homology-directed repair), enables in vivo precise genome editing to insert a sequence encoding an epitope tag or a fluorescent protein into a gene of interest. Although precise genome editing has not been possible in the mammalian brain in vivo due to the lack of homologous recombination activity in post mitotic neurons, we achieved single-cell, precise genome editing by delivering the editing machinery to dividing neuronal progenitors through in utero electroporation. We demonstrated that SLENDR is scalable to many species of proteins in diverse cell types and ages, and permits high-resolution imaging with light and electron microscopy both in fixed and live brain tissue.

Therefore, SLENDR allows researchers to rapidly and precisely determine the localization and dynamics of endogenous proteins with the resolution of micro- to nanometers in various cell types, regions and ages of the brain, providing a new platform suitable for large-scale analysis on a broad spectrum of proteins.

 

Reference

High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by In Vivo Genome Editing. Takayasu Mikuni, Jun Nishiyama, Ye Sun, Naomi Kamasawa, and Ryohei Yasuda. Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.04.044.

Pubmed link