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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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Targeting G-quadruplex Forming Sequences with Cas9.

Hamza Balci1,2, Viktorija Globyte2, Chirlmin Joo2

  • 1Department of Physics, Kent State University, Kent, Ohio 44242, United States.

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|March 26, 2021
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Summary
This summary is machine-generated.

CRISPR-Cas9 gene editing is affected by DNA structures like G-quadruplexes (GQ). This study reveals how GQ formation in target DNA influences CRISPR-Cas9 complex structures and dynamics, impacting its activity.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • CRISPR-Cas9 systems offer precise DNA targeting and editing capabilities.
  • Noncanonical DNA secondary structures, such as G-quadruplexes (GQ), can influence protein-DNA interactions.
  • The impact of GQ formation on CRISPR-Cas9 complex structure and function remains largely uncharacterized.

Purpose of the Study:

  • To investigate the structural characteristics of the CRISPR-Cas9 complex when interacting with target DNA containing G-quadruplex forming sequences (PQS).
  • To assess how the stability and location (target or non-target strand) of PQS influence the CRISPR-Cas9 complex conformation and dynamics.

Main Methods:

  • Single-molecule Förster Resonance Energy Transfer (smFRET) was employed to study the structural dynamics of the CRISPR-Cas9 complex bound to DNA.
  • Target DNA sequences with varying PQS stability and positions were synthesized and analyzed.

Main Results:

  • The conformational states and dynamics of the CRISPR-Cas9 complex varied significantly based on GQ stability and PQS location.
  • When PQS was on the non-target strand (NTS), GQ formation was observed for both weak and stable GQs, facilitated by R-loop formation.
  • When PQS was on the target strand (TS), R-loop formation supported weak GQ formation but not moderate or stable GQs.

Conclusions:

  • Structural heterogeneity within the target DNA and R-loop is strongly dependent on PQS location (TS vs. NTS).
  • These structural variations likely have functional implications for CRISPR-Cas9 activity and editing efficiency.
  • Understanding these structural influences is crucial for optimizing CRISPR-Cas9 applications.