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Genome Editing in Human Pluripotent Stem Cells Using Site-Specific Nucleases

  • Kunitoshi Chiba
  • Dirk HockemeyerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1239)

Abstract

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) (Thomson, Science 282:1145–1147, 1998; Takahashi et al. Cell 131:861–872, 2007), collectively referred to as pluripotent stem cells (hPSCs), are currently used in disease modeling to address questions specific to humans and to complement our insight gained from model organisms (Soldner et al. Cell 146:318–331, 2011; Soldner and Jaenisch, Science 338:1155–1156, 2012). Recently, genetic engineering using site-specific nucleases has been established in hPSCs (Hockemeyer et al. Nat Biotechnol 27:851–857, 2009; Hockemeyer et al., Nat Biotechnol 29:731–734, 2011; Zou et al., Cell Stem Cell 5:97–110, 2011; Yusa et al., Nature 478:391–394, 2011; DeKelver et al., Genome Res 20:1133–1142, 2010), allowing a level of genetic control previously limited to model systems. Thus, we can now perform targeted gene knockouts, generate tissue-specific cell lineage reporters, overexpress genes from a defined locus, and introduce and repair single point mutations in hPSCs. This ability to genetically engineer pluripotent stem cells will significantly facilitate the study of human disease in a defined genetic context. Here we outline protocols for efficient gene targeting in hPSCs.

Key words

Genome editing Site-specific nucleases ZFN TALEN CRISPR/Cas9 Human pluripotent stem cells hESCs hiPSC 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Molecular and Cell BiologyUniversity of California, BerkeleyBerkeleyUSA

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