Related Experiment Video

Updated: Mar 24, 2026

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
09:04

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Published on: September 25, 2019

8.9K

Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells

Susan M Byrne1, George M Church1

  • 1Department of Genetics, Harvard Medical School, Boston, Massachusetts.

Current Protocols in Stem Cell Biology
|March 8, 2016
PubMed

Insights

This study presents an optimized CRISPR/Cas9 protocol for genome engineering in human induced pluripotent stem cells (hiPSCs), achieving high transfection and editing efficiencies without selection. The method enables precise gene modification in challenging cell types.

Area of Science:

  • Molecular Biology
  • Genetics
  • Stem Cell Biology

Background:

  • CRISPR/Cas9 systems enable precise gene editing via targeted DNA double-strand breaks.
  • Human embryonic stem cells and induced pluripotent stem cells (iPSCs) present challenges for transfection and DNA damage resilience compared to tumor cell lines.

Purpose of the Study:

  • To develop and optimize a transient transfection protocol for efficient genome engineering in human iPSCs.
  • To establish reliable methods for gene disruption, insertion, and replacement in hiPSCs.

Main Methods:

  • Utilized transient transfection of plasmids and single-stranded oligonucleotides for CRISPR/Cas9 delivery into hiPSCs.
  • Developed strategies for designing sgRNA target sites and donor vectors.
  • Employed single-cell FACS sorting for iPSC cloning and genotyping for edited cells.

Main Results:

  • Achieved transfection efficiencies exceeding 60% in hiPSCs.
  • Demonstrated gene disruption efficiencies ranging from 1-25%.
  • Obtained gene insertion/replacement efficiencies between 0.5-10% without selection or enrichment.

Conclusions:

  • The optimized protocol significantly enhances the efficiency and precision of genome engineering in human iPSCs.
  • This method provides a robust platform for genetic modification of hiPSCs, facilitating downstream applications in regenerative medicine and disease modeling.
Keywords:
CRISPR / Cas9 nucleasegene targetinggenome engineeringhuman induced pluripotent stem cellstransfection

More Related Videos

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions04:21

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

Published on: November 1, 2024

1.4K
Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation09:51

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Published on: February 2, 2016

14.3K

Related Concept Videos

Induced Pluripotent Stem Cells01:13

Induced Pluripotent Stem Cells

Stem cells are undifferentiated cells that divide and produce different types of cells. Ordinarily, cells that have differentiated into a specific cell type are post-mitotic—that is, they no longer divide. However, scientists have found a way to reprogram these mature cells so that they “de-differentiate” and return to an unspecialized, proliferative state. These cells are also pluripotent like embryonic stem cells—able to produce all cell types—and are therefore...
28.5K
Induced Pluripotent Stem Cells01:06

Induced Pluripotent Stem Cells

Stem cells are undifferentiated cells that divide and produce different cell types. Ordinarily, cells that have differentiated into a specific cell type are terminally differentiated; however, scientists have found a way to reprogram these mature cells so that they dedifferentiate and return to an unspecialized, proliferative state. These cells are pluripotent like embryonic stem cells—able to produce all cell types—and are called induced pluripotent stem cells (iPSCs).
Somatic...
6.2K
Induced Pluripotent Stem Cells01:13

Induced Pluripotent Stem Cells

4.0K
CRISPR01:59

CRISPR

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...
58.9K
CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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...
2.5K
Chromatin Modification in iPS Cells01:32

Chromatin Modification in iPS Cells

Chromatin modification alters gene expression; therefore, scientists can add histone-modifying enzymes, histone variants, and chromatin remodeling complexes to somatic cells to aid reprogramming into pluripotent stem (iPS) cells.
Compact chromatin makes reprogramming difficult. Enzymes, such as histone demethylases and acetyltransferases, are often added during reprogramming to loosen the chromatin, making the DNA more accessible to transcription factors. Molecules that inhibit histone...
2.3K