Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

The generation of a collection of human induced pluripotent stem cell (hiPSC) lines expressing endogenously GFP-tagged proteins using CRISPR/Cas9 methods is described. The methods used and the genomic and cell biological data validating the GFP-tagged hiPSC lines are also presented.
We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in