CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells
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Fluorescent reporter and epitope-tagged human pluripotent stem cells (hPSCs) greatly facilitate studies on the pluripotency and differentiation characteristics of these cells. Unfortunately traditional procedures to generate such lines are hampered by a low targeting efficiency that necessitates a lengthy process of selection followed by the removal of the selection cassette. Here we describe a procedure to generate fluorescent reporter and epitope tagged hPSCs in an efficient one-step process using the CRISPR/Cas technology. Although the method described uses our recently developed iCRISPR platform, the protocols can be adapted for general use with CRISPR/Cas or other engineered nucleases. The transfection procedures described could also be used for additional applications, such as overexpression or lineage tracing studies.
Key wordsHuman pluripotent stem cells (hPSCs) Gene targeting CRISPR/Cas Homologous recombination Knockin Fluorescent reporter Epitope tag
Nipun Verma and Zengrong Zhu contributed equally to this work. Our work related to this publication was funded, in part, by NIH (R01DK096239) and NYSTEM (C029156). Z.Z. was supported by the New York State Stem Cell Science (NYSTEM) fellowship from the Center for Stem Cell Biology (CSCB) of the Sloan Kettering Institute. N.V. was supported by the Howard Hughes Medical Institute (HHMI) Medical Research and the Tri-Institutional Weill Cornell/ Rockefeller/ Sloan Kettering MD-PhD program.