Comprehensive Protocols for CRISPR/Cas9-based Gene Editing in Human Pluripotent Stem Cells

  • Curr Protoc Stem Cell Biol. 2016 Aug 17:38:5B.6.1-5B.6.60. doi: 10.1002/cpsc.15.
David P Santos  1 ,  Evangelos Kiskinis  1 ,  Kevin Eggan  2  3 ,  Florian T Merkle  4
Affiliations
  • 1. The Ken & Ruth Davee Department of Neurology & Clinical Neurological Sciences, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois.
  • 2. Department of Stem Cell and Regenerative Biology, and Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts.
  • 3. Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts.
  • 4. Metabolic Research Laboratories and Medical Research Council Metabolic Diseases Unit, Wellcome Trust-Medical Research Council Institute of Metabolic Science, and Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.
PMID: 27532820 DOI: 10.1002/cpsc.15
Abstract

Genome editing of human pluripotent stem cells (hPSCs) with the CRISPR/Cas9 system has the potential to revolutionize hPSC-based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC culture or the CRISPR/Cas9 system, to successfully perform genome editing. The methods are presented in detail and are supported by a theoretical framework to allow for the incorporation of inevitable improvements in the rapidly evolving gene-editing field. We describe protocols to generate hPSC lines with gene-specific knock-outs, small targeted mutations, or knock-in reporters. © 2016 by John Wiley & Sons, Inc.

Keywords
CRISPR; gene editing; knock-in; pluripotent; stem cell.