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Related Concept Videos

CRISPR01:59

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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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The Nucleosome Core Particle01:12

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Nucleosomes are the DNA-histone complex, where the DNA strand is wound around the histone core. The histone core is an octamer containing two copies of H2A, H2B, H3, and H4 histone proteins.
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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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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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Related Experiment Video

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Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP
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Structural insights into how Cas9 targets nucleosomes.

Reina Nagamura1, Tomoya Kujirai2, Junko Kato2

  • 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

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|December 31, 2024
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CRISPR-Cas9 genome editing targets nucleosomal DNA in eukaryotes. Cas9 preferentially cleaves linker DNA, not tightly wrapped DNA, offering insights for improved chromatin-based gene editing tools.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Structural Biology

Background:

  • CRISPR-associated endonuclease Cas9 is a key genome editing tool.
  • Eukaryotic DNA is packaged into chromatin, with nucleosomes as the basic unit.
  • Understanding Cas9's interaction with nucleosomal DNA is crucial for eukaryotic genome editing.

Purpose of the Study:

  • To investigate the structural basis of Cas9 targeting within nucleosomes.
  • To elucidate how Cas9 interacts with DNA in the context of chromatin.
  • To inform the development of advanced genome editing technologies.

Main Methods:

  • Native-polyacrylamide gel electrophoresis (PAGE) for DNA cleavage analysis.
  • Cryo-electron microscopy (cryo-EM) to determine the structure of Cas9-sgRNA-nucleosome complexes.
  • In vitro and in vivo assays to assess Cas9 activity on nucleosomal DNA.

Main Results:

  • Cas9 targets linker DNA and entry-exit DNA regions of nucleosomes, avoiding tightly wrapped DNA.
  • Cryo-EM reveals multiple interaction sites between Cas9 and nucleosomes.
  • Mutations reducing Cas9-nucleosome interaction enhance in vitro cleavage, with limited in vivo inhibition.

Conclusions:

  • Cas9's interaction with nucleosomes influences its DNA cleavage activity.
  • Structural insights into Cas9-nucleosome binding are vital for understanding its function in chromatin.
  • Findings pave the way for engineering more effective chromatin-based genome editing tools.