Generation of Endogenously Tagged Membrane Trafficking Regulators Using CRISPR Genome Editing.

Vesicle trafficking entails packaging and transport of membrane-associated proteins to their target membranes, and recycling or degradation of endocytosed proteins. Biochemical and cell biological studies of vesicle trafficking often require the introduction of epitope tags or fluorescent protein markers for protein purification and tracking in cells. Previously, such tagging experiments in mammalian cells mainly used overexpression systems, which could lead to artifacts. Abnormally high expression levels also prevent us from studying individual vesicle trafficking events with precision. With the advent of CRISPR technologies, epitope tags and fluorescent proteins can now be introduced into endogenous proteins in almost any cell type that are proliferating in culture. This chapter describes approaches for inserting tags at the endogenous loci of genes, with the vesicle tethering protein complex, exocyst, as an example.