Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Cas9 is a bacterial enzyme used for genome editing in eukaryotic cells.
• Chromatin structure, particularly nucleosomes, can influence DNA-targeting enzymes.
• Understanding these interactions is crucial for improving CRISPR-Cas9 efficiency.

Purpose of the Study:

• To investigate the impact of nucleosome structure on Cas9 endonuclease activity.
• To determine how DNA target location within or near a nucleosome affects Cas9 targeting.
• To identify the critical factors governing Cas9 activity on chromatinized DNA.

Main Methods:

• In vitro characterization of Cas9 activity using purified nucleosome substrates.
• Assays to measure Cas9 DNA cleavage efficiency at various target site positions relative to the nucleosome.
• Analysis of the role of the protospacer-adjacent motif (PAM) accessibility.

Main Results:

• Cas9 endonuclease activity is significantly inhibited when the target DNA sequence is located within the nucleosome core.
• Cas9 activity remains unaffected when the target site is in the linker DNA adjacent to the nucleosome.
• Accessibility of the protospacer-adjacent motif (PAM) is the primary determinant of Cas9 activity on nucleosomal DNA.

Conclusions:

• Nucleosome core particle formation strongly inhibits Cas9 targeting.
• Cas9 can efficiently target DNA in linker regions, suggesting potential for targeting accessible chromatin.
• PAM accessibility is a critical factor for Cas9 function on chromatin, guiding strategies for enhanced genome editing in vivo.