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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

685
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...
685
CRISPR01:59

CRISPR

53.5K
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...
53.5K

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Related Experiment Video

Updated: Oct 22, 2025

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
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Published on: March 12, 2018

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Knock-in Blunt Ligation Utilizing CRISPR/Cas9.

Jonathan M Geisinger1, Michele P Calos2

  • 1Department of Biology, Stanford University, Stanford, USA.

Bio-Protocol
|August 30, 2021
PubMed
Summary
This summary is machine-generated.

CRISPR/Cas9 technology enables precise gene editing by creating DNA breaks for inserting new genetic material. This study details a generalized knock-in blunt ligation protocol applicable to various cell types, including stem cells.

Keywords:
CRISPR/Cas9Cell cultureDouble-strand breaksGenome engineeringNHEJ

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas9 is a powerful tool for genome manipulation.
  • Cas9 generates blunt-ended double-strand DNA breaks.
  • Precise introduction of exogenous DNA is crucial for genetic engineering.

Purpose of the Study:

  • To present a generalized protocol for knock-in blunt ligation.
  • To adapt CRISPR/Cas9 technology for precise DNA insertion.
  • To demonstrate the utility of this method in HEK293 cells.

Main Methods:

  • Utilizing Cas9 to create blunt-ended double-strand breaks.
  • Exploiting non-homologous end-joining DNA repair pathways.
  • Introducing exogenous DNA with high precision.

Main Results:

  • Successfully applied the protocol to immortalized cell lines and human induced pluripotent stem cells.
  • Developed a generalized method for knock-in blunt ligation.
  • Demonstrated the protocol's effectiveness using HEK293 cells as an example.

Conclusions:

  • The presented protocol offers a precise method for gene knock-in using CRISPR/Cas9.
  • This technique expands the capabilities of genome engineering.
  • The generalized protocol is adaptable for various cell types.