The kindest cut may be no cut or, in the case of genome editing, no double-strand break (DSB). Although a DSB in DNA is the usual result when the CRISPR/Cas9 genome-editing system is used, modified versions of CRISPR/Cas9 avoid cutting into the genome and instead manipulate the epigenome. Rather than change genes—and risk introducing potentially harmful mutations—epigenome-targeting CRISPR/Cas9 systems change gene expression.
Such epigenome-targeting CRISPR/Cas9 systems could have therapeutic applications, if only they could demonstrate sufficient safety, practicality, and efficiency. This last quality—efficiency—was emphasized in a recent study completed by Salk Institute researchers. These scientists developed a novel CRISPR/Cas9 system that preserves DNA integrity while activating target genes in mouse models of human disease. According to the scientists, their system ameliorated disease symptoms in mice afflicted with diabetes, muscular dystrophy, or acute kidney disease.
Details of the scientists’ work appeared December 7 in the journal Cell, in an article entitled, “In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-Epigenetic Modulation.” The article describes a epigenome-targeting CRISPR/Cas9 system for gene activation, that is, a system that induces gain-of-function changes in the epigenome.
“Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling,” wrote the article’s authors. “The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs.”
The principal idea behind the Salk technique is the use of two adeno-associated viruses (AAVs) as the machinery to introduce their genetic manipulation machinery to cells in postnatal mice. The researchers inserted the gene for the Cas9 enzyme into one AAV virus, and they used another AAV virus to introduce a short single guide RNA (sgRNA), which specifies the precise location in the mouse genome where Cas9 will bind, and a transcriptional activator.
The shorter sgRNA is only 14 or 15 nucleotides long compared with the standard 20 nucleotides used in most CRISPR/Cas9 techniques. By using a relatively diminutive sgRNA, the Salk team devised a system in which Cas9 was unable to cut DNA.
“Cutting DNA opens the door to introducing new mutations,” said the Cell article’s senior author, Juan Carlos Izpisua Belmonte, Ph.D., a professor at the Salk Institute for Biological Studies. “That is something that is going to stay with us with CRISPR or any other tool we develop that cuts DNA. It is a major bottleneck in the field of genetics—the possibility that the cell, after the DNA is cut, may introduce harmful mistakes.”
In the current study, the Salk team used the modified gRNA to bring a transcriptional activator to work together with the Cas9 enzyme. In this way, the team induced epigenetic remodeling by recruiting transcriptional machinery (a process the scientists call trans-epigenetic modulation) to targeted loci.
Essentially, the transcriptional machinery sits in the region of DNA of interest and promotes expression of a gene of interest. Similar techniques could be used to activate virtually any gene or genetic pathway without the risk of introducing potentially harmful mutations.
“We wanted to change the cell fate with therapeutic efficiency without a DNA cut,” co-first author Fumiyuki Hatanaka explained.
Strikingly, the team demonstrated disease reversal in several disease models in mice. In a mouse model of acute kidney disease, they showed that the technique activated previously damaged or silenced genes to restore normal kidney function. They were also able to induce some liver cells to differentiate into pancreatic-like cells, which produce insulin, to partially rescue a mouse model of type 1 diabetes.
The team also showed that they could recover muscle growth and function in mouse models of muscular dystrophy, a disease with a known gene mutation. Instead of trying to correct the mutated gene, the researchers increased the expression of genes in the same pathway as the mutated gene, over-riding the effect of the damaged gene. “We are not fixing the gene; the mutation is still there,” says Belmonte. “Instead, we are working on the epigenome, and the mice recover the expression of other genes in the same pathway. That is enough to recover the muscle function of these mutant mice.”
Preliminary data suggest that the technique is safe and does not produce unwanted genetic mutations. However, the researchers are pursuing further studies to ensure safety, practicality, and efficiency before considering bringing it to a clinical environment.
Belmonte sees this technology as a way of potentially treating neurological disorders such as Alzheimer's and Parkinson's diseases. Just as the technique restored kidney, muscle, and insulin-producing function in the mouse models, he sees a future for rejuvenating neuronal populations, maybe even one day in human patients.