Tagging endogenous loci for live-cell fluorescence imaging and molecule counting using ZFNs, TALENs, and Cas9

Abstract

The programmable ZFN, TALEN, and Cas9 nucleases allow genome editing of any cell line or organism. In this chapter, we describe methods to create gene fusions at endogenous loci in mammalian cells to express fluorescent fusions of proteins of interest at endogenous levels. The donor DNA, which includes the sequence encoding a fluorescent protein, is provided to the cell to repair a double-strand break induced by a nuclease. The engineered donor sequence is integrated by homology-directed repair into the genome in frame with the coding region of the gene of interest, resulting in expression of a fusion protein at physiological levels. We further describe techniques to study protein dynamics and numbers using the genome-edited cell lines. In contrast to cell lines stably overexpressing fusion proteins from modified cDNAs, genes encoding fluorescent proteins are targeted to the endogenous genetic locus, avoiding perturbation of alternative splicing and expression levels.

Keywords: Donor plasmid; Endogenous; Fluorescence microscopy; GFP; Genome editing; Nuclease.