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CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells
Nipun Verma1,2, Zengrong Zhu1, Danwei Huangfu3
1Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY, 10065, USA.
Methods in Molecular Biology (Clifton, N.J.)
|November 4, 2016
Summary
Generating fluorescent reporter and epitope-tagged human pluripotent stem cells (hPSCs) is now efficient using CRISPR/Cas technology. This one-step method bypasses lengthy selection processes for faster stem cell research.
Area of Science:
- Stem Cell Biology
- Gene Editing Technologies
Background:
- Human pluripotent stem cells (hPSCs) are crucial for studying pluripotency and differentiation.
- Traditional methods for generating tagged hPSCs are inefficient, requiring lengthy selection and cassette removal.
Purpose of the Study:
- To develop an efficient one-step procedure for generating fluorescent reporter and epitope-tagged hPSCs.
- To adapt CRISPR/Cas technology for streamlined stem cell line generation.
Main Methods:
- Utilized CRISPR/Cas technology, specifically the iCRISPR platform, for gene editing in hPSCs.
- Developed a one-step transfection protocol for generating tagged hPSC lines.
Main Results:
- Achieved efficient generation of fluorescent reporter and epitope-tagged hPSCs.
- The described method significantly reduces the time and effort compared to traditional techniques.
Conclusions:
- The novel CRISPR/Cas-based method offers an efficient and streamlined approach to generating engineered hPSCs.
- This protocol can be adapted for various CRISPR/Cas systems and other applications like overexpression and lineage tracing.
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