Nucleosomes impede Cas9 access to DNA in vivo and in vitro

Abstract

The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties.

DOI: http://dx.doi.org/10.7554/eLife.12677.001

eLife digest

Many bacteria have a type of immune system known as CRISPR that can target and cut foreign DNA to protect it against viruses. Recently, the CRISPR system was adapted to allow scientists to easily manipulate the genome of humans and many other organisms. However, unlike the loosely organized DNA found in bacteria, the DNA that makes up the human genome is tightly packed and wrapped around complexes of proteins to form structures called nucleosomes. It was not clear whether the CRISPR system was able to effectively target the stretches of DNA in a nucleosome.

In 2013, researchers developed a modified version of CRISPR, known as CRISPR interference, to block gene activity and in 2014 used it to systematically repress many of the genes in the human genome. Now, Horlbeck, Witkowsky et al. – who include several of the researchers from the 2014 work – have analyzed existing data for a specific type of human cell grown in the laboratory and found that CRISPR interference activity was strongest in certain areas around the start of each gene. However, CRISPR interference was much weaker in other areas of genes that coincided well with stretches of DNA that are known to often be bound by nucleosomes. Nucleosomes also appeared to block CRISPR editing, although the effects were less pronounced.

Horlbeck, Witkowsky et al. then directly tested whether nucleosomes could prevent the CRISPR system from binding or modifying the DNA. When the individual components were mixed in test tubes, the CRISPR system could readily target “naked” DNA. However, it could not access nucleosome-bound DNA, unless an enzyme that can move nucleosomes along the DNA in the human genome was also added to the mix. These findings suggest one way that CRISPR can manipulate much of the human genome despite the widespread presence of nucleosomes. Future work will now aim to develop computational methods that take the positions of nucleosomes into account when picking DNA sites to target with CRISPR.

DOI: http://dx.doi.org/10.7554/eLife.12677.002