Elife. 2016 Apr 28;5. pii: e13450. doi: 10.7554/eLife.13450.

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function.

Isaac RS^1,², Jiang F^3,⁴, Doudna JA^4,^5,^6,^7,⁸, Lim WA^9,^10,¹¹, Narlikar GJ¹, Almeida R^10,^11,¹².

Author information

1: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States.
2: Tetrad Graduate Program, University of California, San Francisco, San Francisco, United States.
3: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
4: California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States.
5: Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States.
6: Department of Chemistry, University of California, Berkeley, Berkeley, United States.
7: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States.
8: Innovative Genomics Initiative, University of California, Berkeley, Berkeley, United States.
9: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.
10: Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, United States.
11: California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, United States.
12: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.

Abstract

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.

KEYWORDS:

ATP-dependent chromatin remodeling; CRISPR; CRISPR-Cas9; biochemistry; chromatin; chromosomes; genes; none; nucleosome

PMID:: 27130520
PMCID:: PMC4880442
DOI:: 10.7554/eLife.13450

[Indexed for MEDLINE]

Free PMC Article

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Figure 1.

Cas9 DNA nuclease activity is hindered by nucleosomes.

(A) Schematic of sgRNAs designed against the assembled 601 80/80 nucleosome substrates targeting the flanking regions, entry/exit sites, and near the nucleosomal dyad. (B) Cleavage assay comparing Cas9 cleavage on 80/80 DNA and 80/80 nucleosomes when loaded with sgRNA #3. (C) Kinetics of cleavage with sgRNA #3. (D) Comparison of the relative rates of cleavage on nucleosomes to DNA at various positions along the 80/80 nucleosome construct. The position reported is the site of cleavage by Cas9. Represented values are mean ± SEM from three replicates.
DOI: http://dx.doi.org/10.7554/eLife.13450.003
10.7554/eLife.13450.004Figure 1—source data 1.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNAs #2 and #6.DOI: http://dx.doi.org/10.7554/eLife.13450.004
10.7554/eLife.13450.005Figure 1—source data 2.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNAs #2 and #6.DOI: http://dx.doi.org/10.7554/eLife.13450.005
10.7554/eLife.13450.006Figure 1—source data 3.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNAs #2 and #6.DOI: http://dx.doi.org/10.7554/eLife.13450.006
10.7554/eLife.13450.007Figure 1—source data 4.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNA #5.DOI: http://dx.doi.org/10.7554/eLife.13450.007
10.7554/eLife.13450.008Figure 1—source data 5.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNA #5.DOI: http://dx.doi.org/10.7554/eLife.13450.008
10.7554/eLife.13450.009Figure 1—source data 6.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNA #1.DOI: http://dx.doi.org/10.7554/eLife.13450.009
10.7554/eLife.13450.010Figure 1—source data 7.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNA #1.DOI: http://dx.doi.org/10.7554/eLife.13450.010
10.7554/eLife.13450.011Figure 1—source data 8.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNA #3.DOI: http://dx.doi.org/10.7554/eLife.13450.011
10.7554/eLife.13450.012Figure 1—source data 9.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNA #3.DOI: http://dx.doi.org/10.7554/eLife.13450.012
10.7554/eLife.13450.013Figure 1—source data 10.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNA #4.DOI: http://dx.doi.org/10.7554/eLife.13450.013
10.7554/eLife.13450.014Figure 1—source data 11.Replicate gels of Cas9 cleavage of 80/80 601 DNA and nucleosomes with sgRNA #4.DOI: http://dx.doi.org/10.7554/eLife.13450.014
10.7554/eLife.13450.015Figure 1—source data 12.Quantification of Cas9 cleavage gels.DOI: http://dx.doi.org/10.7554/eLife.13450.015

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

eLife. 2016;5:e13450.

Figure 2.

Higher nucleosomal breathing dynamics enhance Cas9 cleavage.

(A) Schematic illustrating nucleosome breathing and how it can enable Cas9 binding to a target in the nucleosome. (B) Cleavage assay comparing Cas9 cleavage of 601 and 5S 0/0 nucleosomes when loaded with sgRNAs targeting comparable positions at core and entry sites. (C) Quantification of (B). (D) Cas9 cleavage rates on 601 and 5S nucleosomes when targeted to core and entry sites. Values were normalized against naked DNA control rates. Represented values are mean ± SEM from three replicates. Additional gel panels shown in .
DOI: http://dx.doi.org/10.7554/eLife.13450.021
10.7554/eLife.13450.022Figure 2—source data 1.Replicate gels of cleavage of 0/0 5S DNA and nucleosomes with sgRNA core.DOI: http://dx.doi.org/10.7554/eLife.13450.022
10.7554/eLife.13450.023Figure 2—source data 2.Replicate gels of cleavage of 0/0 5S DNA and nucleosomes with sgRNA core.DOI: http://dx.doi.org/10.7554/eLife.13450.023
10.7554/eLife.13450.024Figure 2—source data 3.Replicate gels of cleavage of 0/0 5S DNA and nucleosomes with sgRNA entry.DOI: http://dx.doi.org/10.7554/eLife.13450.024
10.7554/eLife.13450.025Figure 2—source data 4.Replicate gels of cleavage of 0/0 5S DNA and nucleosomes with sgRNA entry.DOI: http://dx.doi.org/10.7554/eLife.13450.025
10.7554/eLife.13450.026Figure 2—source data 5.Replicate gels of cleavage of 0/0 601 DNA and nucleosomes with sgRNA entry.DOI: http://dx.doi.org/10.7554/eLife.13450.026
10.7554/eLife.13450.027Figure 2—source data 6.Replicate gels of cleavage of 0/0 601 DNA and nucleosomes with sgRNA entry.DOI: http://dx.doi.org/10.7554/eLife.13450.027
10.7554/eLife.13450.028Figure 2—source data 7.Quantification of Cas9 cleavage gels.DOI: http://dx.doi.org/10.7554/eLife.13450.028
10.7554/eLife.13450.029Figure 2—source data 8.Quantification of Cas9 cleavage gels.DOI: http://dx.doi.org/10.7554/eLife.13450.029

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

eLife. 2016;5:e13450.

Figure 3.

Chromatin remodeling improves Cas9 cleavage of nucleosomal substrates.

(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

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

eLife. 2016;5:e13450.