CRISPR software creates long-lasting epigenome alterations | Spectrum
Epigenome editing: A new CRISPR tool modifies chemical tags in the genome that silence gene expression for months, even as cells divide and differentiate.
Keith Chambers / Science Photo Library
A new variant of the classic CRISPR gene editing tool can change the chemical markings of the genome and suppress gene expression for months. Researchers could use the approach to tailor the activity of genes linked to autism.
CRISPR systems typically use an enzyme called Cas9 to cut target DNA sequences and remove or replace them. Editing genes with CRISPR, however, harbors risks: The tool can produce unwanted, permanent changes in the DNA and lead to cell death.
Rather than manipulating DNA directly, researchers can turn genes on or off by changing the epigenome, a series of chemical modifications in the genome that control expression. However, this approach typically requires cells to express a foreign protein for as long as researchers want to suppress or increase expression of a gene, which runs the risk of triggering an immune response.
With the new approach, researchers can permanently change gene expression after only briefly exposing cells to an epigenetic editor called CRISPRoff. The technique described in Cell in April shortens the time cells are exposed to foreign proteins. To reverse the changes, the team developed another tool called CRISPRon, which removes methyl groups from DNA in certain places.
“We can do the same things as CRISPR by being able to permanently turn off genes, but we can do this without mutating the genome,” said Luke Gilbert, assistant professor of urology at the University of California at San Francisco. who led the work.
Gilbert and his colleagues created CRISPRoff with an inactivated version of the Cas9 protein, which no longer cuts DNA, but can still be directed to a target site via strands of RNA. Then they fused Cas9 with two different classes of proteins: one modified labels on proteins called histones that DNA wraps around, and the other added chemical labels known as methyl groups to the DNA. Both changes silence gene expression.
To test the system, the team delivered the protein complex along with RNA segments that target various genes into cultured human cells. In one experiment, they targeted a few selected genes, measured gene expression levels, and logged DNA methylation patterns to look for unintended “off-target” effects. In another experiment, the team used CRISPRoff to silence genes in stem cells and then grow them into neurons.
CRISPRoff silenced target gene expression in the vast majority of cells for at least 50 days, although the protein complex itself disappeared after 10 days, the researchers reported. In some batches of cells, the target gene remained silent for 450 cell divisions or 15 months. And stem cells retained the changes in gene expression as the cells differentiated into neurons.
The researchers also reported that the tool is highly specific. CRISPRoff attenuated the activity of target genes without significantly changing the expression levels of neighboring genes. With CRISPRon, the researchers were able to reactivate expression in more than 70 percent of the cells.
The team also assessed CRISPRoff’s ability to silence a range of genes. They introduced segments of RNA targeting more than 20,000 genes into a pool of cells, with each cell carrying an RNA pair targeting the same gene. As the RNAs are converted to DNA and integrated into a cell’s genome, the researchers grew the cells and sequenced their DNA to measure the frequency of certain sets of RNA.
RNAs targeting genes known to be essential for cell proliferation were depleted, demonstrating the effectiveness of CRISPRoff. The result suggests that CRISPRoff can silence the majority of genes in the genome, the researchers say.
The team was also able to silence genes missing regions known as CpG islands – previously believed to be critical in regulating genes through methylation. However, the results suggest that these genes are just as controllable as genes with CpG islets, says Gilbert.
Researchers could use CRISPRoff and CRISPRon to study how the epigenome affects gene activity and what role it plays in conditions like autism, the researchers say. Ultimately, the tools could be used to develop therapies that regulate gene expression.