title: Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro.
abstract: During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.
authors.affiliation: School of Molecular Biosciences and Center for Reproductive Biology, Washington State University , Pullman, Washington 99164-7520, United States.
authors.forename: John M
authors.initials: JM
authors.lastname: Hinz
authors.affiliation: School of Molecular Biosciences and Center for Reproductive Biology, Washington State University , Pullman, Washington 99164-7520, United States.
authors.forename: Marian F
authors.initials: MF
authors.lastname: Laughery
authors.affiliation: School of Molecular Biosciences and Center for Reproductive Biology, Washington State University , Pullman, Washington 99164-7520, United States.
authors.forename: John J
authors.initials: JJ
authors.lastname: Wyrick
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D004247:DNA / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism*; D013297:Streptococcus pyogenes / Q000201:enzymology*; D014981:Xenopus
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D009707:Nucleosomes; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases