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Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization
- Source :
- Molecular Biology of the Cell
- Publication Year :
- 2017
-
Abstract
- The generation of a collection of human induced pluripotent stem cell (hiPSC) lines expressing endogenously GFP-tagged proteins using CRISPR/Cas9 methods is described. The methods used and the genomic and cell biological data validating the GFP-tagged hiPSC lines are also presented.<br />We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in
- Subjects :
- 0301 basic medicine
Population
Green Fluorescent Proteins
Induced Pluripotent Stem Cells
Fluorescent Antibody Technique
Biology
Green fluorescent protein
Cell Line
03 medical and health sciences
0302 clinical medicine
Genome editing
Myosin
Methods
CRISPR
Beta-actin
Humans
Induced pluripotent stem cell
education
Molecular Biology
030304 developmental biology
Trans-activating crRNA
Fibrillarin
Gene Editing
0303 health sciences
education.field_of_study
Cas9
Stem Cells
Cell Biology
Articles
Molecular biology
3. Good health
Cell biology
030104 developmental biology
Gene Targeting
Nuclear lamina
Stem cell
CRISPR-Cas Systems
030217 neurology & neurosurgery
Subjects
Details
- ISSN :
- 19394586
- Volume :
- 28
- Issue :
- 21
- Database :
- OpenAIRE
- Journal :
- Molecular biology of the cell
- Accession number :
- edsair.doi.dedup.....da0c0c83c01daafcdf4f1b7b369b5ae6