Scientists have discovered how to modify the basic architecture of CRISPR to extend its reach beyond the genome and into what is known as the epigenome – proteins and small molecules that cling to DNA and control when and where genes are turned on or off.
In an article published on April 9, 2021 in the journal Cell, researchers at UC San Francisco and the Whitehead Institute describe a new CRISPR-based tool called “CRISPRoff,” which allows scientists to turn off almost all genes in human cells without making a single change to the genetic code. The researchers also show that once a gene is deactivated, it remains inert in the cell’s descendants for hundreds of generations, unless it is reactivated with a complementary tool called CRISPRon, also described in article.
Because the epigenome plays a central role in many diseases, from viral infection to cancer, CRISPRoff technology could one day lead to powerful epigenetic therapies. And since this approach does not involve any DNA modification, it is probably safer than conventional CRISPR therapies, which are known to cause unwanted and potentially damaging genome changes.
“Although genetic and cell therapies are the future of medicine, there are potential safety concerns associated with permanent genome modification, which is why we are trying to find other ways to use CRISPR to treat disease, ”said Luke Gilbert, PhD, professor at UCSF’s Helen Diller Family Comprehensive Cancer Center and co-lead author of the new article.
Transform CRISPR from genome to epigenome editor
Conventional CRISPR is equipped with two pieces of molecular material that make it an efficient gene editing tool. One of the components is a DNA capture enzyme, which gives CRISPR the ability to modify DNA sequences. The other is a reference device that can be programmed to focus on any DNA sequence of interest, giving precise control over where changes are made.
To construct CRISPRoff, the researchers dispensed with the conventional DNA capture enzyme function of CRISPR while retaining the feedback device, creating a stripped CRISPR capable of targeting any gene, but not modifying it. Then they attached an enzyme to this barebones CRISPR. But rather than splicing DNA, this enzyme acts on the epigenome.
The new tool targets a particular epigenetic characteristic known as DNA methylation, which is one of the many molecular parts of the epigenome. When DNA is methylated, a small chemical tag known as a methyl group is put on the DNA, which silences neighboring genes. Although DNA methylation occurs naturally in all mammalian cells, CRISPRoff offers scientists unprecedented control over this process. Another tool described in the article, called CRISPRon, removes methylation marks deposited by CRISPRoff, making the process fully reversible.
“We now have a simple tool that can silence the vast majority of genes,” said Jonathan Weissman, PhD, fellow of the Whitehead Institute, lead co-author of the new paper and former UCSF faculty member. “We can do this for multiple genes at the same time without any damage to the DNA, and in a way that can be reversed. It’s a great tool for controlling gene expression.”
“Major Surprise” upsets a basic principle of epigenetics
Based on previous work by a group in Italy, the researchers were convinced that CRISPRoff would be able to silence specific genes, but they suspected that around 30% of human genes would not respond to the new tool.
DNA is made up of four genetic letters – A, C, G, T – but, in general, only Cs next to G can be methylated. To complicate matters, scientists have long believed that methylation could only silence genes at sites in the genome where CG sequences are highly concentrated, regions called “CpG islands”.
Since nearly a third of human genes do not have CpG islands, the researchers speculated that methylation would not turn off these genes. But their CRISPRoff experiments turned this epigenetic dogma upside down.
“What was believed prior to this work was that the 30 percent of genes that lack CpG islets were not controlled by DNA methylation,” Gilbert said. “But our work clearly shows that you don’t need a CpG island to turn off genes by methylation. That, to me, was a big surprise.”
Epigenetic inheritance enhances the therapeutic potential of CRISPRoff
Easy-to-use epigenetic editors like CRISPRoff have enormous therapeutic potential, in large part because, like the genome, the epigenome can be inherited.
When CRISPRoff silences a gene, the gene not only remains switched off in the treated cell, but it also remains switched off in the cell’s offspring when it is divided, for up to 450 generations.
To the researchers’ surprise, this was true even in maturing stem cells. Although the transition from stem cell to differentiated adult cell involves significant rewiring of the epigenome, the methylation marks deposited by CRISPRoff were faithfully inherited in a significant fraction of cells that made this transition.
These results suggest that CRISPRoff should only be administered once to have lasting therapeutic effects, making it a promising approach for the treatment of rare genetic diseases – including Marfan syndrome, which affects connective tissue, the Job syndrome, an immune system disorder and some forms. cancer – which are caused by the activity of a single damaged copy of a gene.
The researchers noted that while CRISPRoff is exceptionally promising, more work is needed to realize its full therapeutic potential. Time will tell if CRISPRoff and similar technologies are indeed “the future of medicine”.