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Updated: Feb 3, 2026
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Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
Published on: September 25, 2019
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Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells
Jean Ann Maguire1, Fabian L Cardenas-Diaz2, Paul Gadue1,2,3,4
1Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
Current Protocols in Stem Cell Biology
|October 26, 2018
Summary
This study introduces a new CRISPR-Cas9 method for precise gene editing in human pluripotent stem cells (PSCs). The streamlined protocol enables accurate genetic modifications for disease modeling and therapeutic development.
Area of Science:
- Stem cell biology
- Gene editing technologies
Background:
- Human pluripotent stem cells (PSCs) hold significant promise for basic research and cell-based therapies.
- CRISPR-Cas9 gene editing enhances the potential of PSCs for disease modeling and correction of mutations.
Purpose of the Study:
- To develop a precise and efficient method for introducing targeted genetic modifications into human PSCs using CRISPR-Cas9.
- To overcome the challenge of unintended indel formation on non-targeted alleles during gene editing.
Main Methods:
- Utilized a dual-oligonucleotide strategy, with one oligonucleotide for sequence alteration and another for maintaining the wild-type sequence.
- Streamlined transfection and screening methodologies for efficiency with limited cell numbers and reduced labor.
Main Results:
- Successfully demonstrated a method for precise genetic modification on a single allele in human PSCs.
- Developed an efficient protocol that minimizes labor-intensive clone passaging.
Conclusions:
- The described protocol offers a streamlined and technically simple approach for precise gene editing in human PSCs.
- This method facilitates the generation of valuable tools for modeling human diseases in stem cells.
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