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Related Concept Videos

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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Induced Pluripotent Stem Cells01:13

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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...
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Related Experiment Video

Updated: Jun 4, 2025

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing
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CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells.

Avinash Singh1,2, Swathy Babu1,2, Marcus Phan1,3

  • 1Department of Neurology, University of Minnesota, Twin Cities, Minneapolis, MN, USA.

Bio-Protocol
|December 30, 2024
PubMed
Summary

Enhance CRISPR genome editing in human induced pluripotent stem cells (iPSCs) by inhibiting p53 and using pro-survival molecules. This method boosts cell survival and achieves over 90% homologous recombination efficiency.

Keywords:
CRISPR Cas9Editing efficiencyHomologous recombination rateNucleofectioniPSCs

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

Published on: September 25, 2019

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Area of Science:

  • Molecular Biology
  • Stem Cell Biology
  • Genetic Engineering

Background:

  • CRISPR/Cas9 technology offers powerful genome editing capabilities.
  • Editing human induced pluripotent stem cells (iPSCs) with CRISPR faces challenges in cell survival and efficiency.
  • P53 activation during CRISPR editing can induce apoptosis, hindering success.

Purpose of the Study:

  • To develop a protocol to enhance cell survival and editing efficiency in human iPSCs using CRISPR.
  • To mitigate the apoptotic response triggered by CRISPR editing.
  • To streamline the creation of isogenic cell lines.

Main Methods:

  • Combining p53 inhibition with pro-survival small molecules during CRISPR editing.
  • Utilizing CRISPR/Cas9 technology for targeted genome modification in human iPSCs.
  • Assessing homologous recombination rates and cell survival post-editing.

Main Results:

  • A novel protocol combining p53 inhibition and pro-survival molecules significantly enhances cell survival.
  • Achieved a homologous recombination rate exceeding 90% in human iPSCs.
  • Reduced the overall time for iPSC genome editing to as little as 8 weeks.

Conclusions:

  • The combination of p53 inhibition and pro-survival molecules is an effective strategy for improving CRISPR editing outcomes in iPSCs.
  • This optimized protocol accelerates the generation of genetically modified iPSCs.
  • The findings facilitate the efficient creation of isogenic cell lines for research and therapeutic applications.