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Related Concept Videos

CRISPR01:59

CRISPR

56.2K
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: Dec 9, 2025

Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Methods for Measuring CRISPR/Cas9 DNA Cleavage in Cells.

Christopher R Cromwell1, Juan Jovel2, Basil P Hubbard3

  • 1Department of Pharmacology, University of Alberta, Edmonton, AB, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|September 14, 2020
PubMed
Summary

CRISPR/Cas9 gene editing offers precision but can cause unintended DNA cuts. New methods now allow scientists to accurately measure these on- and off-target effects in cells, improving model reliability and therapeutic safety.

Keywords:
CRISPR/Cas9DNA cleavage specificityGUIDE-seqGene editingGenome engineeringOff-target effectsT7 endonuclease I assayTargeted high-throughput sequencing in cells

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The CRISPR/Cas9 system is a powerful tool for gene editing, enabling precise gene knockouts and knock-ins.
  • A key limitation of CRISPR/Cas9 is its potential for off-target DNA cleavage, especially in large genomes.
  • Assessing CRISPR/Cas9 specificity is crucial for reliable research and safe therapeutic applications.

Purpose of the Study:

  • To present detailed protocols for evaluating the specificity of CRISPR/Cas9 and related gene editing systems in cellular contexts.
  • To enable researchers to measure both on-target and off-target DNA cleavage events accurately.
  • To provide methods for assessing the reliability of CRISPR/Cas9-generated models and the safety of CRISPR-based therapeutics.

Main Methods:

  • Utilizing targeted approaches to analyze known off-target DNA cleavage sites.
  • Employing unbiased approaches to identify off-target cleavage events across the entire genome.
  • Implementing cellular assays to measure CRISPR/Cas9 on- and off-target DNA cleavage.

Main Results:

  • Developed and detailed protocols for comprehensive CRISPR/Cas9 specificity evaluation.
  • Demonstrated the utility of both targeted and unbiased methods for detecting off-target effects.
  • Provided a framework for assessing the accuracy and safety of CRISPR/Cas9 applications.

Conclusions:

  • The presented techniques offer robust methods for evaluating CRISPR/Cas9 specificity in cellular systems.
  • Accurate assessment of on- and off-target cleavage is essential for validating CRISPR-based research models.
  • These protocols contribute to the safe and effective translation of CRISPR/Cas9 technology into clinical settings.