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Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge

John M Hinz1, Marian F Laughery1, John J Wyrick2

  • 1From the School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164.

The Journal of Biological Chemistry
|October 21, 2016
PubMed

Insights

Nucleosomes can enhance CRISPR-Cas9 gene editing specificity. They inhibit Cas9 activity at off-target DNA sites, especially when mismatches occur in the sgRNA seed region near the nucleosome edge.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Nucleosomes influence Cas9 binding and activity at on-target sites.
  • The effect of nucleosomes on Cas9 activity at off-target sites remains largely unknown.

Purpose of the Study:

  • To investigate the impact of nucleosomes on Cas9 cleavage efficiency at off-target DNA sites in vitro.
  • To determine if nucleosomes can enhance the specificity of Cas9 gene editing.

Main Methods:

  • Utilized a single guide RNA (sgRNA) known to direct Cas9 cleavage at the edge of a positioned 601 nucleosome.
  • Compared Cas9 cleavage of naked DNA substrates versus nucleosome-bound DNA substrates with single-site mismatches.

Main Results:

  • Single mismatches between sgRNA and DNA had minimal impact on Cas9 cleavage of naked DNA.
  • Mismatches strongly inhibited Cas9 cleavage of nucleosome substrates, particularly in the sgRNA seed region.
  • Nucleosomes significantly reduced Cas9 cleavage at off-target sites located at the nucleosome edge.

Conclusions:

  • Nucleosomes can act as a barrier to Cas9 cleavage at off-target sites.
  • Nucleosome positioning may enhance Cas9 specificity by preventing cleavage of mismatched sequences at the nucleosome edge.
Keywords:
CRISPR/CasPAMchromatinendonucleasehistonemismatchnucleosomeoff-target

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