NEW YORK (GenomeWeb) – To get the best results with CRISPR/Cas9, researchers should target genomic loci with fewer nucleosomes, according to a study published last week in eLife.
Scientists from the University of California, Berkeley and the University of California, San Francisco described how the most active guide RNAs (gRNAs) targeted genomic regions with low nucleosome occupancy.
"In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access," the authors wrote. "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."
The findings could inform applications like CRISPR interference (CRISPRi), which requires persistent DNA binding to knock down gene expression, and epigenomic editing, which might take place in genomic regions with higher or static nucleosome occupancy.
Because CRISPR systems evolved in archea and bacteria, they are "likely not optimized to explore and modify large, chromatin-bound eukaryotic genomes," the authors said, adding that several studies have suggested a correlation between DNase I hypersensitivity and the ability of Cas9 to bind and cleave DNA.
The idea to test the hypothesis that nucleosomes impede Cas9 activity came by looking at large data sets from more than 30 gRNA tiling and genome-scale genetic screens, Max Horlbeck, first author and a member of co-senior author Jonathan Weissman's lab at UCSF, told GenomeWeb. Those data sets came not only from the Weissman lab, but also Martin Kampmann's lab, also at UCSF, and Jodi Nunnari's lab at UC-Davis.
"A pattern emerged where [CRISPR] would be very active, then the signal would go away, and then it would come back," Horlbeck said.
Looking at both nuclease activity and decreased gene expression from nuclease-null dCas9, they found that high nucleosome occupancy was "strongly depleted of highly active sgRNAs."
Their findings were similar to those of co-senior author Robert Tijan, of UC-Berkeley, who found with in vitro CRISPR experiments that nucleosomes blocked Cas9 activity.
"It became very clear that what we were seeing was a reflection of this fundamental block," Horlbeck said.
Moreover, in follow-up experiments the scientists were able to boost Cas9 activity in vitro by adding a chromatin remodeling enzyme, yChd1.
The results of these experiments could have implications for several applications of CRISPR/Cas9 technology.
"For genome editing, where all you need is a single cleavage event for any given target site, this will be less important," Horlbeck said. "And while it might fuel a new generation of [guide RNA] library design, with the dynamic nature of the nucleosome landscape it won't be as strong of an effect."
Using Cas9 to make a genomic edit might be able to get around the nucleosomes, because it only needs to succeed in cutting DNA once, and "could potentially take advantage of nucleosome turnover during replication."
But scientists using dCas9 to inhibit gene expression or Cas9 to make edits to epigenomic regions of the genome would be wise to consider this new study, Horlbeck said.
The results will be "very important for any application that uses dCas9 where you need long-term binding," he said, such as gene knock down using CRISPRi. And for those looking to edit regulatory regions such as transcription start sites, where the nucleosome landscape is static compared to protein-coding regions, researchers would be advised to avoid regions with high nucleosome occupancy.
The findings could also "provide a new dimension for selecting highly active gRNAs," they wrote, especially for CRISPRi. Early work on gRNA sequences provided algorithms for calculating how efficiently a given gRNA might lead Cas9 to the target and how often it might lead Cas9 astray, to similar off-target loci. Incorporating the new study might improve the algorithm's ability to find targets within a particular gene for knock down.
How much attention should be paid to nucleosome inhibition in future studies? "It depends a lot on the application and the [genomic] region," Horlbeck said.