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

By Brock Roberts, Amanda Haupt, Andrew Tucker, Tanya Grancharova, Joy Arakaki, Margaret A. Fuqua, Angelique Nelson, Caroline Hookway, Susan A. Ludmann, Irina A Mueller, Ruian Yang, Alan R. Horwitz, Susanne M. Rafelski, Ruwanthi N. Gunawardane

Posted 31 Mar 2017
bioRxiv DOI: 10.1101/123042 (published DOI: 10.1091/mbc.E17-03-0209)

We present a CRISPR/Cas9 genome editing strategy to systematically tag endogenous proteins with fluorescent tags in human inducible pluripotent stem cells. To date we have generated multiple human iPSC lines with GFP tags for 10 proteins representing key cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, lamin B1, non-muscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome editing methodology using Cas9 ribonuclear protein electroporation and fluorescence-based enrichment of edited cells resulted in <0.1-24% HDR across all experiments. Clones were generated from each edited population and screened for precise editing. ~25% of the clones contained precise mono-allelic edits at the targeted locus. Furthermore, 92% (36/39) of expanded clonal lines satisfied key quality control criteria including genomic stability, appropriate expression and localization of the tagged protein, and pluripotency. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. The data described here, including our editing protocol, genetic screening, quality control assays, and imaging observations, can serve as an initial resource for genome editing in cell biology and stem cell research.

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