Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

R Stefan Isaac; Fuguo Jiang; Jennifer A Doudna; Wendell A Lim; Geeta J Narlikar; Ricardo Almeida

doi:10.7554/eLife.13450

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

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

Authors

R Stefan Isaac^{1

2}, Fuguo Jiang^{3

4}, Jennifer A Doudna^{4

5

6

7

8}, Wendell A Lim^{9

10

11}, Geeta J Narlikar¹, Ricardo Almeida^{10

11

12}

Affiliations

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.

MeSH terms

Animals
Bacterial Proteins / metabolism*
CRISPR-Associated Protein 9
CRISPR-Cas Systems*
DNA / metabolism*
Endonucleases / metabolism*
Gene Editing / methods*
Nucleosomes / metabolism*
Protein Binding
Recombination, Genetic*
Xenopus

Substances

Bacterial Proteins
Nucleosomes
DNA
CRISPR-Associated Protein 9
Cas9 endonuclease Streptococcus pyogenes
Endonucleases

Abstract

MeSH terms

Substances

Grants and funding