Title | CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells. |
Publication Type | Journal Article |
Year of Publication | 2017 |
Authors | Verma N, Zhu Z, Huangfu D |
Journal | Methods Mol Biol |
Volume | 1513 |
Pagination | 119-140 |
Date Published | 2017 |
ISSN | 1940-6029 |
Keywords | Bacterial Proteins, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Doxycycline, Endonucleases, Epitopes, Gene Knock-In Techniques, Genes, Reporter, Genetic Loci, Genetic Vectors, Green Fluorescent Proteins, Homologous Recombination, Humans, Lipids, Luminescent Proteins, Pluripotent Stem Cells, RNA, Guide, Transfection |
Abstract | 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. |
DOI | 10.1007/978-1-4939-6539-7_9 |
Alternate Journal | Methods Mol. Biol. |
PubMed ID | 27807834 |
PubMed Central ID | PMC5554709 |
Grant List | P30 CA008748 / CA / NCI NIH HHS / United States R01 DK096239 / DK / NIDDK NIH HHS / United States / HHMI / Howard Hughes Medical Institute / United States |
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