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CRISPR01:59

CRISPR

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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In-vitro Mutagenesis01:16

In-vitro Mutagenesis

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To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
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Related Experiment Video

Updated: Apr 7, 2026

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions
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CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

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Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9.

Yuejun Chen1, Jingyuan Cao1, Man Xiong2

  • 1Waisman Center, University of Wisconsin, Madison, WI 53705, USA.

Cell Stem Cell
|July 7, 2015
PubMed
Summary
This summary is machine-generated.

Researchers developed a new method to create inducible gene knockout (iKO) human pluripotent stem cell (hPSC) lines. This technique enables precise control over gene deletion, advancing the study of gene function in human cells.

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Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9
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Area of Science:

  • Stem Cell Biology
  • Molecular Genetics
  • Gene Editing Technologies

Background:

  • Precise temporal control of gene expression or deletion is crucial for understanding gene function.
  • Establishing human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) is technically challenging.

Purpose of the Study:

  • To develop an efficient strategy for generating iKO hPSC lines.
  • To enable precise temporal control of gene deletion in hPSCs for functional studies.

Main Methods:

  • Combined CRISPR/Cas9 genome editing with Flp/FRT and Cre/LoxP systems.
  • Utilized a dual-sgRNA targeting approach for biallelic knockin of FRT sequences.
  • Developed a two-step strategy for simultaneous insertion of a recombinase cassette and removal of drug-resistance genes.

Main Results:

  • Successfully established iKO hPSC lines (hESC and iPSC) for multiple genes (SOX2, PAX6, OTX2, AGO2).
  • Demonstrated the efficiency of the dual-sgRNA targeting and two-step strategy.
  • Generated iKO lines for genes with diverse structural and expression patterns.

Conclusions:

  • The developed strategy significantly speeds up the generation of iKO hPSC lines.
  • Availability of these iKO hPSC lines will transform gene function analysis in human cells.
  • This method provides a powerful tool for studying gene function in developmental and disease contexts.