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Related Concept Videos

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Nucleosomes are the basic units of chromatin compaction. Each nucleosome consists of the DNA bound tightly around a histone core, which makes the DNA inaccessible to DNA binding proteins such as DNA polymerase and RNA polymerase. Hence, the fundamental problem is to ensure access to DNA when appropriate, despite the compact and protective chromatin structure.
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DNA in a human cell is almost 2m long and it is packed inside a tiny nucleus that is only a few microns in diameter. The level of compaction of DNA inside the nucleus is astonishing. It is organized into several sequentially higher levels of compaction to fit into such a tiny space. The most compact form of DNA is a chromosome that can be seen under a microscope in a dividing cell.
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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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Nucleosome breathing and remodeling constrain CRISPR-Cas9 function.

R Stefan Isaac1,2, Fuguo Jiang3,4, Jennifer A Doudna4,5,6,7,8

  • 1Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States.

Elife
|May 1, 2016
PubMed
Summary
This summary is machine-generated.

CRISPR-Cas9 genome editing is effective in eukaryotic cells, even within nucleosomes. DNA sequence and proximity to the nucleosome dyad affect accessibility, while chromatin remodelers enhance Cas9 activity.

Keywords:
ATP-dependent chromatin remodelingCRISPRCRISPR-Cas9biochemistrychromatinchromosomesgenesnonenucleosome

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Area of Science:

  • Molecular Biology
  • Epigenetics
  • Genome Engineering

Background:

  • The CRISPR-Cas9 system is a powerful tool for genome editing in eukaryotic cells.
  • Understanding how CRISPR-Cas9 interacts with eukaryotic chromatin is crucial for its effective application.
  • Nucleosomes, the basic units of chromatin, pose potential barriers to CRISPR-Cas9 access.

Purpose of the Study:

  • To investigate how nucleosomes constrain the activity of the CRISPR-Cas9 system.
  • To determine the factors influencing CRISPR-Cas9 accessibility to nucleosomal DNA.
  • To explore the role of chromatin remodeling enzymes in facilitating CRISPR-Cas9 action on chromatin.

Main Methods:

  • In vitro assembly of nucleosomes on native DNA sequences.
  • Assays to measure Cas9 accessibility and activity on nucleosomal DNA.
  • Investigating the impact of DNA sequence dynamics and PAM site location relative to the nucleosome dyad.
  • Testing the effect of chromatin remodeling enzymes on Cas9 activity.

Main Results:

  • Nucleosomes are generally permissive to CRISPR-Cas9 action.
  • Cas9 accessibility to nucleosomal DNA varies significantly based on DNA sequence properties and PAM site position.
  • Chromatin remodeling enzymes demonstrably enhance Cas9 activity on nucleosomal templates.
  • The dynamic nature of nucleosomal DNA and the action of remodelers are key factors for in vivo chromatin targeting.

Conclusions:

  • CRISPR-Cas9 can overcome nucleosomal barriers in eukaryotic cells.
  • Nucleosomal DNA accessibility is modulated by intrinsic DNA properties and chromatin organization.
  • Chromatin remodeling enzymes play a significant role in optimizing CRISPR-Cas9 efficacy on chromatin.
  • These findings provide insights into the in vivo mechanisms governing CRISPR-Cas9 chromatin targeting.