(A) Schematic of the experimental setup. Supercoiled plasmid containing the 601 sequence inserted into a pBlueScript II SK (+) backbone (pBSIISK+601) was chromatinized by step gradient salt dialysis in the presence of histone octamer. Purified yeast Chd1 (yChd1) remodeling factor was used to test the effect of ATP-dependent remodeling factors on Cas9 access to nucleosomal DNA. (B) Quality assessment of the chromatinized plasmid used in this study. Titrated amounts of Micrococcal Nuclease (MNase) were incubated with the chromatinized plasmid, and the resulting pattern of protection by assembled nucleosomes was visualized on a 1.3% agarose gel post-stained with ethidium bromide (EtBr). As a control, the supercoiled plasmid was also incubated with the lowest concentration of MNase. (C) A restriction enzyme accessibility assay (REAA) was used to assess the occupancy and position of the nucleosome assembled at the 601 sequence within the chromatinized plasmid. A panel of unique restriction enzyme sites spanning the 601 sequence were incubated with either the supercoiled plasmid, or the chromatinized plasmid. Cleavage was stopped, and protein was removed by incubation with proteinase K followed by Phenol:Chloroform:Isoamyl alcohol extraction and ethanol precipitation. (Top) The resulting DNA was then linearized using DraIII, and the level of cleavage by the restriction enzyme panel was visualized on a 1% agarose gel post-stained with EtBr. The label 'P' represents supercoiled plasmid, while 'C' represents chromatinized plasmid. (Bottom right) The location of the restriction sites used are indicated on a diagram of the plasmid. (Bottom left) After quantification of the gel, the percent protection from cleavage experienced in the chromatinized plasmid was plotted versus the location of the cleavage sites on the top strand of the 601 sequence. Experiment 1 refers to the REAA experiment shown in the gel above, while experiment 2 refers to the REAA experiment without remodeler shown in Figure 5F. The grey shading indicates the borders of the 601 sequence, and the grey oval represents the corresponding nucleosome. (D) REAA experiment using the frequent cutter, HaeIII, to assess the remodeling activity around the chromatinized plasmid by the purified yChd1 chromatin remodeler. The resulting banding patterns were visualized on a 1.5% agarose gel post-stained with EtBr. Low molecular weight fragments indicate a high degree of HaeIII accessibility, while higher weight bands indicate protection from digestion. (E) Diagram showing the location of the restriction enzyme cleavage sites and the PAMs targeted by Cas9/sgRNA in the experiment shown in (F) and (G). (F) An accessibility assay was performed essentially as in C using either restriction enzymes or Cas9/sgRNAs in the presence or absence of the remodeler yChd1. The level of cleavage by the restriction enzyme panel (left) or Cas9/sgRNAs (right) was visualized on a 1.3% agarose gel post-stained with EtBr. A negative control was conducted with an sgRNA that had no sequence complementarity to the plasmid used (non-sense guide). The concentration of yChd1 used was the same as in panel (D) (G) Quantification of the gels shown in F. Percent protection from cleavage of the chromatinized plasmid in the presence or absence of the chromatin remodeler was calculated relative to the percent cleavage in the corresponding supercoiled plasmid control, and plotted at the location of the restriction enzyme cleavage sites or the center of the PAMs with respect to the 601 dyad.