Whether CRISPR is used for genomic editing or epigenomic manipulation, it must demonstrate a high degree of specificity—the ability to be “on target” far more often than not. In the genome-editing case, when CRISPR is teamed with a nuclease-active Cas9 enzyme to snip at selected DNA locations, off-target effects are relatively easy to assess. In the epigenome-manipulation case, when CRISPR is teamed with a nuclease-inactive Cas9 enzyme and a transcription-modifying protein, off-target effects are less, well, clear-cut. Accordingly, the potential for epigenome editing with CRISPR systems has been difficult to assess.
Resolving the issue of whether CRISPR can serve as an effective epigenome-manipulation tool is a matter of some urgency. If CRISPR could control the genome’s switches with a high degree of specificity, it would likely advance the study and treatment of human diseases that are driven by mutations in control regions of the genome. Such diseases include cancer, cardiovascular disorders, neurodegenerative conditions, and diabetes.
CRISPR, it turns out, is capable of precisely controlling gene expression. That, at least, is the conclusion reached by scientists at Duke University. These scientists conducted gene-silencing experiments with a CRISPR system in which a nuclease-inactive dCas9 was fused to the Krüppel-associated box (KRAB) repressor. Then they assessed the degree to which the resulting dCas9-KRAB demonstrated genome-wide specificity, as well as the extent to which it catalyzed heterochromatin formation.
The results appeared October 26 in the journal Nature, in an article entitled, “Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements.”
“We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets,” wrote the authors. “Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression.”
The authors asserted that their result demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome. “[Our] experiments show exceptional specificity, demonstrating that the technology is capable of targeting single sequences of the genome,” said Charles A. Gersbach, Ph.D., associate professor of biomedical engineering at Duke.
“Looking at the specificity of these tools is technically very challenging,” said Timothy E. Reddy, Ph.D., assistant professor of biostatistics and bioinformatics at Duke. “Finding a change in sequence or gene activity is relatively straightforward if you're focusing on one concentrated area in the genome. But looking at how turning off one enhancer switch affects the activity and structure of the whole genome requires more specialized techniques.”
Dr. Gersbach turned to Dr. Reddy and colleague Gregory E. Crawford, Ph.D., who all work together in adjacent laboratories and offices in Duke's Center for Genomic and Computational Biology, for help with these more specialized techniques.
“Current methods for controlling gene switches, including drugs used in clinical trials, change the activity of many switches across the genome simultaneously, creating thousands of off-target effects,” said Dr. Crawford. “What is desperately needed is a technology for manipulating one element at a time.”
It fell to Pratiksha Thakore, a Ph.D. student in Dr. Gersbach's lab, to integrate the expertise of all three laboratories for studying the specificity of CRISPR in controlling these switches. While the results can't prove that every experiment will have the same high level of precision, it provides a blueprint for researchers to assess these effects.
The researchers demonstrated targeting dCas9-KRAB to the HS2 enhancer disrupted the expression of multiple globin genes. Then researchers conducted a genome-wide analysis of dCas9-KRAB binding and repression activity. This analysis, the researchers decided, established that RNA-guided synthetic repressors are highly specific for endogenous target loci. Finally, the researchers concluded that deposition of H3K9me3 was limited to the intended HS2 region, leading to decreased chromatin accessibility at both the targeted enhancer and its associated promoters.
“By integrating genomics and genome engineering, we have developed a method to comprehensively interrogate how this genetic silencing system works and also suggested ways the technology can be used in the future,” commented Thakore. “We also learned some new things about gene regulation, and it turned out that in this context CRISPR can achieve a much higher level of specificity than we expected.”