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Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
Published on: August 25, 2018
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Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
Amanda Haupt1, Tanya Grancharova1, Joy Arakaki1
1Allen Institute for Cell Science.
Journal of Visualized Experiments : Jove
|September 11, 2018
Summary
This study presents a CRISPR/Cas9 protocol for fluorescently tagging proteins in human induced pluripotent stem cells (hiPSCs). This method allows for studying protein dynamics under natural gene regulation in various cell types.
Area of Science:
- Molecular Biology
- Stem Cell Biology
- Gene Editing
Background:
- Studying endogenous protein function requires precise genetic modification.
- Conventional overexpression systems lack physiological relevance.
- Human induced pluripotent stem cells (hiPSCs) offer a valuable model for studying gene function in diploid, nontransformed cells.
Purpose of the Study:
- To develop a protocol for generating hiPSCs with endogenously tagged proteins.
- To enable the study of protein localization and dynamics under endogenous control.
- To facilitate the investigation of tagged proteins in various isogenic cellular contexts.
Main Methods:
- Utilized the CRISPR/Cas9 system for homology-directed repair (HDR) to introduce fluorescent tags.
- Employed a ribonucleoprotein (RNP)-based approach with electroporation for delivering Cas9 protein, guide RNA (gRNA), and donor template.
- Enriched edited cells using fluorescence-activated cell sorting (FACS) and generated clonal lines for analysis.
Main Results:
- Successfully generated hiPSCs expressing fluorescently tagged endogenous proteins.
- Demonstrated the capability to study tagged proteins under endogenous regulatory control.
- Established a method for analyzing precise gene editing outcomes in clonal cell lines.
Conclusions:
- This protocol provides a robust method for endogenous gene tagging in hiPSCs.
- The approach allows for studying protein behavior in a physiologically relevant manner.
- The generated hiPSC lines serve as a versatile platform for diverse cellular studies.
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