Generation of Monoclonal iPSC Lines with Stable Cas9 Expression and High Cas9 Activity.

CRISPR/Cas9-mediated gene editing has been rapidly and widely applied in many organisms for delicate genetic manipulation, including human-induced pluripotent stem cells (iPSCs). Gene editing in human iPSCs is promising for genetics and biomedical research due to that gene-edited iPSC still possesses the potential to be differentiated into any cell lineages. In many cases, the generation of Cas9 expressing cell lines is a prerequisite toward performing successful editing of multiplex genes of interest. Here, we describe a simple, effective method to generate stable Cas9 expressing human iPSCs with high Cas9 activity. In this method, stable Cas9 expressing monoclonal human iPSC lines were generated through lentiviral transduction of Cas9 cassette, followed by blasticidin selection and subcloning with low seeding density. After colonies isolation and expansion, a BFP-GFP reporter assay was applied to validate the Cas9 activities of multiple monoclonal lines by flow cytometry (FACS). These Cas9 expressing human iPSCs generated by our method are single cell-derived monoclonal lines with homogenous population and Cas9 activity of up to 99%.