Objective

The combination of two powerful technologies, human induced pluripotent stem cells (hiPSCs) and precise, footprint-free editing using CRISPR/Cas9, allows for a new level of sophistication in the development of disease models. The ability to create hiPS cell lines from donors with disease-specific mutations and to edit mutations into specific genomic backgrounds will enable discoveries with a new level of granularity. Despite progress in improving nuclease specificity and reducing off-target activity with precise tools like CRISPR/Cas9, a major challenge for successful gene editing in hiPSCs is the lack of culture systems that allow researchers to isolate single hiPSCs with the desired mutations and to generate stable, healthy, clonal lines from edited cells. Traditionally, hiPS cells are grown and passaged as colonies. In order to obtain single cells for cloning, the colonies must first be dissociated into a single-cell suspension, which often results in cell death or premature differentiation. Furthermore, gene editing protocols often subject stem cells to harsh conditions that compromise their health and survival. Using the Cellartis® DEF-CS™ 500 Culture System, we can culture hiPS cells in a monolayer with a very high rate of single-cell survival and clone expansion. We used this culture system to develop a complete workflow, starting with CRISPR/Cas9-mediated editing, using Cas9/sgRNA ribonucleoprotein (RNP) complexes delivered into hiPS cells via either