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Related Concept Videos

CRISPR01:59

CRISPR

55.6K
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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Related Experiment Video

Updated: Nov 30, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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iGUIDE Method for CRISPR Off-Target Detection.

Christopher L Nobles1

  • 1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. chris.l.nobles@gmail.com.

Methods in Molecular Biology (Clifton, N.J.)
|November 12, 2020
PubMed
Summary

Genome editing tools can cause unintended DNA mutations. New methods like GUIDE-seq and iGUIDE detect these off-target effects, ensuring safer applications for gene editing technologies.

Keywords:
CRISPRDNA double-strand breaksOff-target analysisSpecificity

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • Genome editing technologies offer revolutionary therapeutic potential.
  • Nonspecific or off-target activity poses a significant risk, potentially causing harmful genomic abnormalities.
  • Accurate identification of off-target mutations is crucial for the safe clinical translation of gene editing.

Purpose of the Study:

  • To present unbiased methods for identifying off-target DNA double-strand breaks induced by genome editing technologies.
  • To enable researchers to pinpoint unintended mutations for improved safety and efficacy.

Main Methods:

  • Utilized oligo-based marker methods, specifically GUIDE-seq and iGUIDE.
  • Detected DNA double-strand breaks across the genome in an unbiased manner.
  • Compared observed breaks with targeted sequences (e.g., guide RNAs) to identify on- and off-target sites.

Main Results:

  • Successfully identified regions of off-target DNA double-strand breaks.
  • Demonstrated the ability to distinguish between intended on-target and unintended off-target modifications.
  • Provided a reliable approach for assessing the specificity of targeted-nuclease technologies.

Conclusions:

  • GUIDE-seq and iGUIDE are effective tools for unbiased off-target mutation detection in genome editing.
  • These methods are essential for validating the safety and specificity of gene editing tools.
  • Advancing the clinical application of genome editing requires rigorous identification and mitigation of off-target effects.