Best known as a gene-editing system, CRISPR/Cas9 is also being used to edit the epigenome, turning on specific gene promoters and enhancers. The trick is to silence CRISPR/Cas9’s DNA-cutting mechanism. Instead, the CRISPR/Cas9 machinery is used to deliver an enzyme, an acetyltransferase, which adds artificial epigenetic marks to the DNA packaging proteins known as histones.
Gene-editing technologies have been used in several investigations of transcriptional regulation, but with mixed results. For example, some technologies intended for transcriptional control do not enzymatically modulate the chromatin state. They remodel the epigenome indirectly, and so they do not allow specific epigenetic markers to be evaluated.
A more direct and more potent approach has been described by researchers from Duke University. They report that they have designed a fusion protein of a nuclease null deactivated Cas9 (dCas9) with the catalytic histone acetyltransferase core domain of the human E1A-associated protein p300, a highly conserved acetyltransferase involved in a wide range of cellular processes. They reasoned that recruitment of an acetyltransferase by dCas9 to a genomic target site would directly modulate epigenetic structure.
The Duke scientists introduced their epigenome-editing system April 6 in Nature Biotechnology, in an article entitled, “Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.” The article’s authors indicated that their fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and proximal and distal enhancers.
“Targeting of the acetyltransferase shows that gene activation is highly specific,” the authors wrote in a prepublication version of their article. “In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with only one guide RNA.”
The scientists, led by Charles Gersbach, Ph.D., assistant professor of biomedical engineering at Duke, noted that their epigenome-editing technology will advance several lines of investigation. It will help researchers explore the roles that particular promoters and enhancers play in cell fate or the risk for genetic disease. In addition, it could provide a new avenue for gene therapies and guiding stem cell differentiation.
“There are already drugs that will affect enhancers across the whole genome, but that's like scorching the earth,” said Timothy Reddy, Ph.D., assistant professor of biostatistics and bioinformatics at Duke. “I wanted to develop tools to go in and modify very specific epigenetic marks in very specific places to find out what individual enhancers are doing.”
Dr. Reddy found that specificity by teaming up with Dr. Gersbach, his neighbor within Duke's Center for Genomic and Computational Biology. “It's like we use CRISPR to find a genetic address so that we can alter the DNA's packaging at that specific site,” said Dr. Reddy.
Dr. Gersbach and Dr. Reddy put their artificial epigenetic agent to the test by targeting a few well-studied gene promoters and enhancers. While these histone modifications have long been associated with gene activity, it wasn't clear if they were enough to turn genes on. And though Dr. Gersbach and Dr. Reddy had previously used other technologies to activate gene promoters, they had not successfully activated enhancers.
To the duo's great surprise, not only did the agent activate the gene promoters, it turned on the adjacent genes better than their previous methods. Equally surprising was that it worked on enhancers as well: they could turn on a gene—or even families of genes—by targeting enhancers at distant locations in the genome, something that their previous gene activators could not do.
But the real excitement from their results is an emerging ability to probe millions of potential enhancers in a way never before possible.
“Some genetic diseases are straightforward—if you have a mutation within a particular gene, then you have the disease,” said Isaac Hilton, Ph.D., a fellow in the Gersbach Lab and first author of the study. “But many diseases, like cancer, cardiovascular disease, or neurodegenerative conditions, have a much more complex genetic component. Many different variations in the genome sequence can affect your risk of disease, and this genetic variation can occur in these enhancers that Tim has identified, where they can change the levels of gene expression. With this technology, we can explore what exactly it is that they're doing and how it relates to disease or response to drug therapies.”
Dr. Gersbach added, “Not only can you start to answer those questions, but you might be able to use this technique for gene therapy to activate genes that have been abnormally silenced or to control the paths that stem cells take toward becoming different types of cells. These are all directions we will be pursuing in the future.”