CRISPR/Cas9‐based Targeted Genome Editing for the Development of Monogenic Diseases Models with Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a formidable tool for disease modeling, drug discovery, and regenerative medicine using human cells and tissues in vitro . Evolving techniques of targeted genome editing, specifically the CRISPR/Cas9 system, allow for the generation of cell lines bearing gene‐specific knock‐outs, knock‐in reporters, and precise mutations. However, there are increasing concerns related to the transfection efficiency, cell viability, and maintenance of pluripotency provided by genome‐editing techniques. The procedure presented here employs transient antibiotic selection that overcomes reduced transfection efficiency, avoids cytotoxic flow sorting for increased viability, and generates multiple genome‐edited pluripotent hPSC lines expanded from a single parent cell. Avoidance of xenogeneic contamination from feeder cells and reduced operator workload, owing to single‐cell passaging rather than clump passaging, are additional benefits. The outlined methods may enable researchers with limited means and technical experience to create human stem cell lines containing desired gene‐specific mutations. © 2018 by John Wiley & Sons, Inc.