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Related Concept Videos

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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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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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: Sep 15, 2025

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
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A CRISPR-Cas9-based tool for dose-dependent DNA damage detection.

Valentyn Oksenych1, Pavlo Petakh2, Denis Kainov1

  • 1Department of Clinical and Molecular Medicine (IKOM), Norwegian University of Science and Technology, Trondheim, Norway.

The FEBS Journal
|July 13, 2025
PubMed
Summary

Researchers created a CRISPR-Cas9 system in yeast to precisely control DNA double-strand breaks. This tool precisely studied DNA damage response and revealed Tel1 kinase localization, offering a scalable platform for genome stability research.

Keywords:
ATCRISPR‐Cas9DNA double‐strand breaksNHEJTel1homologous recombination

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA double-strand breaks (DSBs) are critical DNA lesions that trigger complex cellular responses.
  • Understanding the DNA damage response (DDR) is crucial for fields ranging from cancer research to aging.
  • Existing methods for inducing DSBs often lack precise control over break frequency and location.

Purpose of the Study:

  • To develop a novel CRISPR-Cas9-based system for precise and dose-dependent induction of DSBs in Saccharomyces cerevisiae.
  • To investigate the dynamics and localization of key DDR proteins, such as Tel1 kinase, following controlled DSB induction.
  • To establish a scalable platform for studying genome stability and DDR mechanisms across different organisms.

Main Methods:

  • Utilized a CRISPR-Cas9 system targeting Ty retrotransposons in yeast for sequence-specific DSB induction.
  • Controlled the number of induced DSBs (×1, ×15, or ×59) to enable dose-dependent studies.
  • Employed microscopy techniques to observe the localization and foci formation of the Tel1 kinase in response to DSBs.

Main Results:

  • The developed system allowed for precise, dose-dependent induction of DSBs, facilitating detailed DDR studies.
  • Tel1 kinase was observed to localize to the nuclear periphery and form multiple foci upon DSB induction.
  • The study identified limitations in Cas9 availability at higher break induction levels, providing insights for system optimization.

Conclusions:

  • The novel CRISPR-Cas9 system offers a powerful and scalable tool for dissecting DNA damage response pathways.
  • Precise control over DSB induction enhances the ability to study DDR dynamics and protein localization.
  • This platform has broad applicability for investigating genome stability and DDR mechanisms in various model organisms.