title: Comprehensive Protocols for CRISPR/Cas9-based Gene Editing in Human Pluripotent Stem Cells.
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.
authors.affiliation: The Ken & Ruth Davee Department of Neurology & Clinical Neurological Sciences, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois.
authors.forename: David P
authors.initials: DP
authors.lastname: Santos
authors.affiliation: The Ken & Ruth Davee Department of Neurology & Clinical Neurological Sciences, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois.
authors.forename: Evangelos
authors.initials: E
authors.lastname: Kiskinis
authors.affiliation: Department of Stem Cell and Regenerative Biology, and Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts.|Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts.
authors.forename: Kevin
authors.initials: K
authors.lastname: Eggan
authors.affiliation: 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.
authors.forename: Florian T
authors.initials: FT
authors.lastname: Merkle
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D018929:Cell Culture Techniques; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular; D003114:Colony-Forming Units Assay; D004247:DNA / Q000302:isolation & purification; D005784:Gene Amplification; D000072669:Gene Editing / Q000379:methods*; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000378:metabolism; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D015203:Reproducibility of Results; D014158:Transcription, Genetic
chemical_list: D009841:Oligonucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA
keywords: CRISPR; gene editing; knock-in; pluripotent; stem cell