Journal article
CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells
Current protocols in stem cell biology, Vol.44(1), pp.5B.7.1-5B.7.22
02/28/2018
DOI: 10.1002/cpsc.46
PMCID: PMC5846340
PMID: 29512106
Abstract
Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR-based genome editing of patient-specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR-based genome editing of patient- specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre-lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc.
Details
- Title: Subtitle
- CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells
- Creators
- Joseph C Giacalone - Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IowaTasneem P Sharma - Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IowaErin R Burnight - Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IowaJohn F Fingert - Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IowaRobert F Mullins - Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IowaEdwin M Stone - Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IowaBudd A Tucker - Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa
- Resource Type
- Journal article
- Publication Details
- Current protocols in stem cell biology, Vol.44(1), pp.5B.7.1-5B.7.22
- DOI
- 10.1002/cpsc.46
- PMID
- 29512106
- PMCID
- PMC5846340
- NLM abbreviation
- Curr Protoc Stem Cell Biol
- ISSN
- 1941-7322
- eISSN
- 1938-8969
- Publisher
- United States
- Grant note
- R01 EY024605 / NEI NIH HHS P30 EY025580 / NEI NIH HHS R01 EY026008 / NEI NIH HHS R01 EY024588 / NEI NIH HHS T32 GM007337 / NIGMS NIH HHS
- Language
- English
- Date published
- 02/28/2018
- Academic Unit
- The University of Iowa Institute for Vision Research; Iowa Neuroscience Institute; Ophthalmology and Visual Sciences
- Record Identifier
- 9983980078202771
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