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Duke Scientists Manipulate Epigenome with CRISPR/Cas9

NEW YORK (GenomeWeb) – No longer just a genomic editing tool, CRISPR/Cas9 can also edit the epigenome, according to a study published today in Nature Biotechnology.

By fusing a non-cutting dCas9 protein to a histone-modifying protein, scientists from Duke University, led by Isaac Hilton and Charles Gersbach, created a system that could selectively raise levels of gene expression by increasing activity at a variety of regulatory regions. Moreover, it was able to do so at high specificity with only a single guide RNA.

Specifically, they took the core protein domain of the human histone acetyltransferase p300 and attached it to the RNA-guided dCas9 protein. The scientists were able to lop off extraneous amino acids in p300 to create a protein focused solely on histone acetylation, which they called the p300 core. The resulting fusion, dubbed dCas9p300core, initiated significantly higher levels of gene expression than existing CRISPR/Cas9-based systems did when targeting epigenomic regions, according to the authors.

They said their method was the first to describe targeted histone acetylation as a way to control gene expression and that it was the first CRISPR/Cas9-based system for epigenome editing.

While projects such as ENCODE have identified millions of epigenetic marks across the human genome, studying the function of those marks "has been largely limited to determining statistical associations with gene expression," the authors wrote.

Existing complexes fusing an activation domain, such as VP64, to dCas9 can increase gene activation, but have some limitations for manipulating the epigenome. They may require multiple activation domains or combinations of gRNAs to achieve high levels of gene induction and do not modify the chromatin state.

By programming the dCas9p300core protein complex to target various epigenetic marks, the scientists were able to induce gene expression from a variety of regulatory regions, including a distal regulatory region, a core enhancer region, proximal and distal enhancer regions, and a locus control region that orchestrated transcription of multiple downstream genes.

The dCas9p300core system not only showed high specificity and low off-target effects, in several cases it worked just as well when directed by a single guide RNA. It also was able to modify chromatin structure through its inherent acetyltransferase activity.

The authors said that the p300 core domain could be attached to other genome editing nucleases, including transcription activator-like effectors and zinc finger nucleases, suggesting a broad range of possibilities for uncovering the workings of the epigenome.

"The unique activity of dCas9p300 core supports its use in elucidating key steps of gene regulation, including dissection of the interplay between the epigenome, regulatory element activity, and gene regulation," they said, adding that it could be combined with light-inducible or chemically inducible proteins that would "enable dynamic control of gene activation in space and time."

The epigenome editing complex could enable other researchers to perform genome-wide screens of regulatory elements -- even targeting multiple elements at once, allowing scientists to manipulate the epigenome to control cell phenotype, the researchers said.