Fig. 4: Cas9-core DNA interaction affect nucleosome DNA cleavage efficiency in vitro.

a Close-up view of the suggested interactions between the PI domain and nucleosomal core DNA. The lysine (K), arginine (R) and histidine (H) residues that potentially contact the DNA are highlighted. b In vitro binding activities of wild-type Cas9 and Cas9 mutants reducing interaction between Cas9 and core DNA for nucleosomes. The nucleosome was incubated with the Cas9-sgRNA complex at 37 °C for 10 min. The Cy3 fluorescence of nucleosomes in the reaction product was visualized by Native-PAGE analysis, and quantified by ImageJ. Data are mean ± s.d. (n = 3 biologically independent samples). The experiments were repeated three times with similar results. c In vitro nucleosome DNA cleavage activities of wild-type Cas9 and mutants reducing interaction between Cas9 and core DNA. The nucleosome was incubated with the Cas9-sgRNA complex at 37 °C for 30 min. The 6-FAM fluorescence of nucleosomes in the reaction product was visualized by Native-PAGE analysis, and quantified by ImageJ. Data are mean ± s.d. (n = 3 biologically independent samples). The experiments were repeated three times with similar results. t tests, * P < 0.05, ** P < 0.01. d Schematic of the experimental setup for in vitro binding and cleavage assays. e In vivo mutation efficiencies at three target sites (SPL7, SPL17 and GAPDH) in transgenic rice calli of the wild-type Cas9 and Cas9 mutants reducing interaction between Cas9 and core DNA for nucleosomes. Dots indicate the mutation frequency for each independent calli. Bars indicate the average mutation frequency. Error bars indicate the standard error (SE). (The number of calli used for each WT, H1264D/R1298Q, H1264E/R1298Q and H1264A/R1298Q was SPL17; n = 14, 17, 17, 17: SPL17; n = 15, 17, 17, 17: GAPDH; n = 15, 17, 17, 17).