(A) Schematic of Cas9 cleavage assay with remodeling. Cas9 is presented with 601 nucleosomes either untreated or previously remodeled with SNF2h or RSC remodelers. (B) Assay comparing cleavage on untreated and remodeled 80/0 nucleosomes when Cas9 is targeted to exit site (depicted in green). These asymmetric nucleosomes are recentered by SNF2h, exposing the exit target site to Cas9 (C) Quantification of (B). (D) Cleavage rates of 80/0 nucleosomes by Cas9 relative to naked DNA, in the presence or absence of SNF2h. SNF2h improves Cas9 cleavage to ~35% of the naked DNA cleavage rate. (E) Assay comparing Cas9-mediated cleavage at entry site of 80/80 symmetric 601 nucleosomes, either untreated or previously treated with RSC remodeler. RSC can destabilize nucleosome structure and reposition nucleosomes towards the DNA ends. (F) Quantification of (E) (G) Comparison of the rates of cleavage of nucleosomes normalized to DNA control with and without the action of RSC chromatin remodeler. Mean enhancement rates of Cas9 activity by chromatin remodeling are shown. (H) Cleavage rates of 80/80 nucleosomes by Cas9 relative to naked DNA, in the presence or absence of RSC. Cas9 cleavage is substantially enhanced by RSC, attaining ~63% of the naked DNA cleavage rate. Represented values are mean ± SEM from three replicates. Additional gel panels shown in . (I) Model of Cas9 activity in vivo in eukaryotes. Left, stable and strongly positioned nucleosomes impede Cas9 activity (downward arrows). However, nucleosomes in vivo are generally more dynamic (breathing), allowing Cas9 opportunities to target underlying DNA (center). Cas9 accessibility to nucleosomal DNA can be further enhanced by the activity of chromatin remodelers that destabilize and/or reposition nucleosomes (right).
DOI: http://dx.doi.org/10.7554/eLife.13450.031
10.7554/eLife.13450.032Figure 3—source data 1.Replicate gels of cleavage of 80/0 DNA and nucleosomes using sgRNA #4 with or without prior remodeling by Snf2h.DOI: http://dx.doi.org/10.7554/eLife.13450.032
10.7554/eLife.13450.033Figure 3—source Data 2.Replicate gels of cleavage of 80/0 DNA and nucleosomes using sgRNA #4 with or without prior remodeling by Snf2h.DOI: http://dx.doi.org/10.7554/eLife.13450.033
10.7554/eLife.13450.034Figure 3—source data 3.Replicate gels of cleavage of 80/0 DNA and nucleosomes using sgRNA #4 with or without prior remodeling by Snf2h.DOI: http://dx.doi.org/10.7554/eLife.13450.034
10.7554/eLife.13450.035Figure 3—source data 4.Quantification of Cas9 cleavage gels from –.DOI: http://dx.doi.org/10.7554/eLife.13450.035
10.7554/eLife.13450.036Figure 3—source data 5.Replicate gels of cleavage of 80/80 DNA and nucleosomes using sgRNA 601_2 with or without prior remodeling by RSC.DOI: http://dx.doi.org/10.7554/eLife.13450.036
10.7554/eLife.13450.037Figure 3—source data 6.Replicate gels of cleavage of 80/80 DNA and nucleosomes using sgRNA 601_2 with or without prior remodeling by RSC.DOI: http://dx.doi.org/10.7554/eLife.13450.037
10.7554/eLife.13450.038Figure 3—source data 7.Replicate gels of cleavage of 80/80 DNA and nucleosomes using sgRNA 601_2 with or without prior remodeling by RSC.DOI: http://dx.doi.org/10.7554/eLife.13450.038
10.7554/eLife.13450.039Figure 3—source data 8.Quantification of Cas9 cleavage gels from –.DOI: http://dx.doi.org/10.7554/eLife.13450.039