title: Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge.
abstract: Nucleosomes affect Cas9 binding and activity at on-target sites, but their impact at off-target sites is unknown. To investigate how nucleosomes affect Cas9 cleavage at off-target sites in vitro, we used a single guide RNA (sgRNA) that has been previously shown to efficiently direct Cas9 cleavage at the edge of the strongly positioned 601 nucleosome. Our data indicate that single mismatches between the sgRNA and DNA target have relatively little effect on Cas9 cleavage of naked DNA substrates, but strongly inhibit cleavage of nucleosome substrates, particularly when the mismatch is in the sgRNA "seed" region. These findings indicate that nucleosomes may enhance Cas9 specificity by inhibiting cleavage of off-target sites at the nucleosome edge.
authors.affiliation: From the School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164.
authors.forename: John M
authors.initials: JM
authors.lastname: Hinz
authors.affiliation: From the School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164.
authors.forename: Marian F
authors.initials: MF
authors.lastname: Laughery
authors.affiliation: From the School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164 jwyrick@vetmed.wsu.edu.
authors.forename: John J
authors.identifier: http://orcid.org/0000-0002-7911-3803
authors.initials: JJ
authors.lastname: Wyrick
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D000076987:CRISPR-Associated Protein 9; D002460:Cell Line; D004247:DNA / Q000737:chemistry*; D004720:Endonucleases / Q000737:chemistry*; D009707:Nucleosomes / Q000737:chemistry*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D009707:Nucleosomes; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR/Cas; PAM; chromatin; endonuclease; histone; mismatch; nucleosome; off-target