New spin on CRISPR uses proteins as an on-off switch for gene editing

April 9, 2021

Researchers have developed a switch to turn genes on and off. (Wikimedia Commons/NIH Image Gallery)

U.S. researchers have developed a nondestructive gene-editing protein that acts as a simple off switch for genes, recreating the benefits of the widely used CRISPR-Cas9 system without permanently damaging cells' genetic material.

The method, detailed in a study published April 9 in Cell, has been named CRISPRoffUnlike traditional CRISPR, this method of gene editing is reversible, and because it does not damage the underlying DNA, introduced changes can even be passed down to future lines of cells.

"The big story here is we now have a simple tool that can silence the vast majority of genes," said co-senior author Jonathan Weissman, a professor of medicine at the University of California, San Francisco. "We can do this for multiple genes at the same time without any DNA damage, with a great deal of homogeneity, and in a way that can be reversed. It's a great tool for controlling gene expression."

Although CRISPR-Cas9 made headlines in 2020 when its two inventors won the Nobel Prize in chemistry, it first emerged in the mid-2000s. It has since revolutionized the way scientists study nearly every area of biology, from major areas such as medicine, evolution and agriculture to more unusual projects, such as bringing back extinct animals.   

"Cas-9 you can think of as a molecular scissors that snips DNA, and then the host cell repairs DNA following these cuts," co-senior author Luke Gilbert, an assistant professor of urology at UCSF, said in an interview with The Academic Times. "But that's a very roundabout way to just turn a gene off by mutating the genome. So what we do is, rather than mutating the genome, we just directly turn genes on and off by controlling the epigenetic code."

This code refers to the way individual genes may be silenced or activated based on small chemical modifications in the DNA. The most frequent of these modifications is methylation, the addition of small chemical tags to DNA. When these tags are added, the enzymes that start the process of converting a gene into a protein can no longer access the DNA, effectively shutting the gene down without destroying it entirely.

Together with researchers from Stanford University, Chan Zuckerberg Biohub and Massachusetts General Hospital and Harvard Medical School, Gilbert and Weissman tapped into this epigenetic code with their new CRISPRoff protein, which they call a "programmable epigenetic memory writer." The protein acts like a little methylation machine, turning off genes. The process can be reversed with a similar protein called CRISPRon, though CRISPRon has not been as carefully studied.

While the applications of CRISPRoff are still in their infancy, the method should theoretically be able to achieve the same results as traditional CRISPR-Cas9. However, it comes with added benefits because it is nondestructive.

"Cells freak out when you destroy the genome," Gilbert said. "When you start chopping up the genome, normal cells recognize that and in many cases will kill themselves in response to damage. And so this approach is nice, in that it doesn't activate those DNA damage responses that are induced by Cas9."

While Gilbert explained that the researchers have only completed "a very tiny fraction of the experiments that one could do with these reagents," the method showed promising results in human cells. The team silenced a gene in stem cells and then induced those stem cells to turn into neurons.

The researchers found that 90% of the neurons kept that gene silent, demonstrating that the gene-silencing from CRISPRoff persists even as cells change type. To explore therapeutic applications, they also silenced genes involved in the production of tau proteins, which are associated with Alzheimer's disease.

The team hopes that by making its work accessible to other researchers, the method will become more widely used and will contribute to better outcomes for CRISPR-based studies.

"It literally is being presented as a resource for other labs to use, and we've put all the reagents that would be needed for folks to use this on a gene, so it's all freely available for academic users," Gilbert said. "It's like open source, essentially, and we hope that people use this widely."

And while it will indeed require many more experiments to discover the full range of possibilities for CRISPRoff and CRISPRon, the researchers have high hopes for their technology.

"We expect that this is a general approach that should be useful in any human cell type — but that's an expectation, not a fact," Gilbert said.

The study, "Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing," published April 9 in Cell, was authored by James K. Nuñez, Jin Chen, Greg C. Pommier, J. Zachery Cogan, Joseph M. Replogle, Gokul N. Ramadoss, Avi J. Samelson, Angela N. Pogson, Amanda Chung, Howard Y. Chang, Martin Kampmann, Luke A. Gilbert and Jonathan S. Weissman, University of California, San Francisco; Carmen Adriaens, Bradley E. Bernstein and Volker Hovestadt, Massachusetts General Hospital and Harvard Medical School; Quanming Shi and King L. Hung, Stanford University; and James Y. S. Kim and Manuel D. Leonetti, Chan Zuckerberg Biohub.

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