Structural insights into how Cas9 targets nucleosomes

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.