CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells

Abstract

Fluorescent reporter and epitope-tagged human pluripotent stem cells (hPSCs) greatly facilitate studies on the pluripotency and differentiation characteristics of these cells. Unfortunately traditional procedures to generate such lines are hampered by a low targeting efficiency that necessitates a lengthy process of selection followed by the removal of the selection cassette. Here we describe a procedure to generate fluorescent reporter and epitope tagged hPSCs in an efficient one-step process using the CRISPR/Cas technology. Although the method described uses our recently developed iCRISPR platform, the protocols can be adapted for general use with CRISPR/Cas or other engineered nucleases. The transfection procedures described could also be used for additional applications, such as overexpression or lineage tracing studies.

Keywords: CRISPR/Cas; Epitope tag; Fluorescent reporter; Gene targeting; Homologous recombination; Human pluripotent stem cells (hPSCs); Knockin.