Fig. 3: Suggested interactions between Cas9 and nucleosome.

a Close-up view of the suggested Cas9-histone tail interacting regions. The cryo-EM density enclosed by a dashed oval indicates the EM density of the histone tail. b In vitro binding activities of the wild-type Cas9 for canonical nucleosomes and tail-less nucleosomes. The nucleosome was incubated with the Cas9-sgRNA complex at 37 °C for 10 min. The Cy5 fluorescence of Cas9 in reaction products were visualized by Native-PAGE analysis with an Amersham Imager 680 (Cytiva), and quantified by ImageJ. Data are mean ± s.d. (n = 3). The experiments were repeated three times with similar results. c In vitro nucleosome DNA cleavage activities of the wild-type Cas9 for canonical nucleosomes and tail-less nucleosomes. The nucleosome was incubated with the Cas9-sgRNA complex at 37 °C for 10 min. The EtBr stained reaction products were visualized by Native-PAGE analysis with an Amersham Imager 680 (Cytiva), and quantified by ImageJ. Data are mean ± s.d. (n = 3). The experiments were repeated three times with similar results. (d) Close-up view of the suggested interacting regions of the PI edge and nucleosomal DNA. The lysine (K) residues that potentially contact the DNA are highlighted. e In vitro binding activities of the wild-type Cas9 and Cas9 mutants to reduce interaction between PI edge and nucleosome DNA entry. The Cy3 fluorescence of nucleosomes in reaction products were visualized by Native-PAGE analysis with an Amersham Imager 680 (Cytiva), and quantified by ImageJ. Data are mean ± s.d. (n = 3). The experiments were repeated three times with similar results. f In vitro nucleosome DNA cleavage activities of the wild-type Cas9 and Cas9 mutants to reduce interaction between PI edge and nucleosome DNA entry. The 6-FAM fluorescence of nucleosomes in reaction products were visualized by Native-PAGE analysis with an Amersham Imager 680 (Cytiva), and quantified by ImageJ. Data are mean ± s.d. (n = 3). The experiments were repeated three times with similar results.