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.
Highlights
The recent development of CRISPR systems, the type II CRISPR-Cas9 mechanism from Streptomyces pyogenes, as an artificial tool for genome engineering, gene regulation, and live imaging is a remarkable achievement with profound impact in a wide variety of research fields and applications (Makarova et al, 2015; Doudna and Charpentier, 2014; Cong et al, 2013; Jinek et al, 2012; 2013; Mali et al, 2013)
We find that the combination of nucleosome breathing, by which DNA transiently disengages from the histone octamer, and the action of chromatin remodeling enzymes allow Cas9 to act on nucleosomal DNA with rates comparable to naked DNA
Recent work using nucleosomes assembled on the 601 sequence has led to the qualitatively similar conclusion that nucleosomes are refractory for Cas9 action (Hinz et al, 2015; Horlbeck et al, 2016)
Summary
The recent development of CRISPR (clustered regularly interspaced short palindromic repeats) systems, the type II CRISPR-Cas mechanism from Streptomyces pyogenes, as an artificial tool for genome engineering, gene regulation, and live imaging is a remarkable achievement with profound impact in a wide variety of research fields and applications (Makarova et al, 2015; Doudna and Charpentier, 2014; Cong et al, 2013; Jinek et al, 2012; 2013; Mali et al, 2013). Bacteria use the CRISPR-Cas system to defend themselves against viruses.
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