CRISPR/Cas9-Mediated Gene Tagging: A Step-by-Step Protocol.

CRISPR/Cas9 provides a simple and powerful tool for modifying almost any DNA of interest. One promising application of the CRISPR/Cas9 system is for tagging genes with a fluorescence marker or tag peptides. For such a purpose, FLAG, HIS, and HA tags or fluorescence proteins (EGFP, BFP, RFP, etc.) have been broadly used to tag endogenous genes of interest. The advantages of generating fluorescence tagging proteins are to provide easy tracing of the subcellular locations, real-time monitoring the expression and dynamics of the protein in different conditions, which cannot be achieved using traditional immunostaining or biochemistry assays. However, the generation of such a gene-tagged cell line could be technically challenging. In this chapter, we demonstrate the generation of tagging the porcine GAPDH (pGAPDH) gene GFP by CRISPR/Cas9-based homology-directed repair.