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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

176
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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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 and crRNAs02:53

CRISPR and crRNAs

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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Homologous Recombination02:31

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Related Experiment Video

Updated: Aug 29, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
08:23

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

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CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy.

Matthew D Newton1,2, Benjamin J Taylor3, Maria Emanuela Cuomo3

  • 1Department of Infectious Disease, Faculty of Medicine, Imperial College London, London, UK.

Methods in Molecular Biology (Clifton, N.J.)
|September 5, 2022
PubMed
Summary
This summary is machine-generated.

CRISPR/Cas9 gene editing offers therapeutic promise but faces challenges from off-target effects. This study uses optical tweezers and microscopy to analyze Cas9 specificity, aiding safer gene therapies.

Keywords:
CRISPRCas9Confocal microscopyFluorescenceForce spectroscopyGene editingOptical tweezers

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetic Engineering

Background:

  • CRISPR/Cas9 is a programmable endonuclease enabling genetic manipulation.
  • Potential for novel gene therapies to treat genetic diseases.
  • Off-target editing by CRISPR/Cas9 presents a significant safety concern for therapeutic applications.

Purpose of the Study:

  • To investigate factors influencing Cas9 specificity.
  • To develop methods for analyzing on- and off-target Cas9 activity.
  • To enhance the safety and efficiency of CRISPR/Cas9-based therapeutics.

Main Methods:

  • Utilized optical tweezers for precise manipulation.
  • Employed confocal fluorescence microscopy for visualization.
  • Integrated microfluidics for controlled experimental conditions.
  • Analyzed both on-target and off-target Cas9 activity.

Main Results:

  • Established a novel methodology for assessing Cas9 specificity.
  • Provided insights into the biophysical parameters governing Cas9 binding and cleavage.
  • Quantified on- and off-target editing events with high resolution.

Conclusions:

  • The developed methods are crucial for understanding and improving Cas9 specificity.
  • This research contributes to the development of safer and more effective gene therapies.
  • Further investigation into Cas9 specificity is vital for clinical translation.