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.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Cell Culture Techniques
  • Cell Line
  • Clone Cells
  • Cloning, Molecular
  • Colony-Forming Units Assay
  • DNA / isolation & purification
  • Gene Amplification
  • Gene Editing / methods*
  • Gene Knockout Techniques
  • Gene Targeting
  • Genes, Reporter
  • Genetic Vectors / metabolism
  • Genome, Human
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Mutation / genetics
  • Oligonucleotides / metabolism
  • Plasmids / genetics
  • Pluripotent Stem Cells / metabolism*
  • Polymerase Chain Reaction
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Reproducibility of Results
  • Transcription, Genetic

Substances

  • Oligonucleotides
  • RNA, Guide, CRISPR-Cas Systems
  • DNA