RESUMO
The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed. Here, we describe a protocol that provides a straightforward method for introducing a stable, highly expressed fluorescent protein into iPSCs using the CRISPR/Cas9 system and a standardized donor vector. The approach involves the integration of the EGFP transgene into the transcriptionally active adeno-associated virus integration site 1 (AAVS1) locus through homology directed repair. The knockin of this transgene results in the generation of iPSC lines with constitutive expression of the EGFP protein that also persists in differentiated iPSCs. These EGFP-labeled iPSC lines are ideal for assessing iPSC differentiation in vitro and evaluating the distribution of iPSC-derived cells in vivo after transplantation into model animals.