Epigenetic Editing with CRISPR Might Be Easier Than We Thought

Researchers at the University of California San Francisco (UCSF) and the Whitehead Institute have developed a novel CRISPR-based tool called “CRISPRoff” that can switch off genes in human cells through epigenetic editing without altering the genetic sequence itself. The tool targets the epigenome, the collection of DNA modifications in a cell such as methylation and post-translational histone modifications. These modifications regulate gene expression without changing the sequence or structure of DNA.

The epigenome plays a key role in many diseases such as heart disease, viral infections and cancer, and the new CRISPRoff technology could lead to powerful epigenetic therapies. The tool could also prove to be safer than conventional CRISPR-based gene therapies as it does not involve DNA editing, and thus would not cause potentially harmful off-target genomic changes.

Dubbed a programmable “epigenetic memory writer,” CRISPRoff initiates heritable gene silencing and once a gene is switched off, it remains silenced in the cell’s progeny for hundreds of generations. Genes can be switched on with a complementary tool called CRISPRon that has also been described in the paper.

The research was published earlier this month in the journal Cell.

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The CRISPR gene editing system consists of the Cas9 enzyme, which serves as molecular scissors to cleave double-stranded DNA, and a guide RNA template targeted to a specific genomic sequence, which allows for precise editing.

Epigenetic Editing with CRISPR

To develop CRISPRoff, the researchers generated a single “dead” Cas9 fusion protein capable of mediating DNA methylation and repressive histone modifications, but devoid of any DNA cleavage function. DNA methylation is an important epigenetic feature where a methyl group is added to DNA bases, namely cytosine in CpG islands (CGIs) in genomic sequences, to silence genes.

DNA methylation occurs endogenously in all mammalian cells in response to various stimuli. While epigenetic therapies such as DNA methyltransferase (DNMT) inhibitors like azacitadine are used as hypomethylating agents in the treatment of diseases like myelodysplastic syndrome and sometimes in AML, CRISPRoff is the first molecular-based epigenetic tool that could modulate the epigenome to treat disease. In the paper, the scientists also describe the CRISPRon “antidote,” which removes methylation marks placed by CRISPRoff, making the process fully reversible.

“Now we have a simple tool that can silence the vast majority of genes,” said Jonathan Weissman, PhD, Whitehead Institute member, co-senior author of the new paper and a former UCSF faculty member in a statement from UCSF. “We can do this for multiple genes at the same time without any DNA damage, and in a way that can be reversed. It’s a great tool for controlling gene expression.”

The researchers tweaked original prototypes of the tool, including those from work by researchers in Italy, to make the CRISPRoff-V1 programmable epigenome editor containing the KRAB domain of the zinc finger transcriptional repressor ZNF10 and (D3A) and two DNMT3 family members, DNMT3A and DNMT3L, fused to catalytically inactive S. pyogenes dCas9.

A surprising finding of the work was that genes without CGIs could also be turned off by methylation. This thwarts the persisting dogma that methylation occurs on “Cs next to Gs” exclusively within CGIs, with the researchers observing that Cs next to Gs outside of CGIs could also be methylated by CRISPRoff. Nearly one third of human genes lack CGIs, which would limit the use of the tool.

Using genome-wide CRISPRoff screens (assessing for proliferation and survival), the scientists identified single guide RNAs (sgRNAs) that could silence the large majority of genes including those lacking canonical annotated CGIs, opening up a much wider targeting window, beyond the limited array of CGI-containing genes, than previously thought.

“What was thought before this work was that the 30 percent of genes that do not have CpG islands were not controlled by DNA methylation,” said Luke Gilbert, PhD, a professor at UCSF’s Helen Diller Family Comprehensive Cancer Center and co-senior author on the paper. Gilbert. “But our work clearly shows that you don’t require a CpG island to turn genes off by methylation. That, to me, was a major surprise.”

Heritable Epigenomic Edits

The researchers also found that CRISPRoff-mediated silencing of a gene ensures that it remains off in the descendants of the cell as it divides for as many as 450 generations (about 15 months). Even more surprising was that this was also true in maturing stem cells. They found that as cells transitioned from induced pluripotent stem cells (iPSCs) to differentiated adult neurons, which involves significant epigenetic changes, the methylation marks made by CRISPRoff were inherited in a significant fraction of cells.

These results suggest that CRISPRoff would potentially only need to be given as a one-time therapy for the treatment of rare genetic diseases, immune system disorders and some forms of cancers that are caused by a faulty copy of a gene.

According to the researchers, the ability of CRISPRoff to induce heritable gene silencing in a broad, heritable fashion, even outside of CGIs, “expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing and mechanistic exploration of epigenetic inheritance.”

The researchers say that although CRISPRoff is exceptionally promising, further work is needed to realize its full therapeutic potential.