Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9.

ABSTRACT: Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescence microscopy of fixed neurons confirmed the identical localization patterns of the tagged proteins to those of endogenous ones verified by immunohistochemistry. The system is also applicable for mult

SUBMITTER: Matsuda T 

PROVIDER: S-EPMC6789158 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature