New CRISPR method acts as an on-off switch for gene expression. Credit: Jennifer Cook-Chrysos/Whitehead Institute
CRISPR-Cas9 gene editing technology has become ubiquitous in the world of biotechnology, and has greatly expanded scientists’ ability to modify the genome at a specific, selectable site. Having just won the 2020 Nobel Prize in Chemistry, it seems CRISPR has been having its moment for the past decade. Despite this, CRISPR edits still come with caveats. One of these is the Cas9 proteins permanent effect on the genome, meaning scientists lack the ability to turn genes on and off reversibly.
In a paper recently published in Cell, a team of researchers detail a form of CRISPR that works around this editing problem. CRISPRoff, designed by Whitehead Institute Member Jonathan Weissman, PhD, University of California San Francisco assistant professor Luke Gilbert, PhD, Weissman lab postdoc James Nuñez, PhD, and collaborators, is fully reversible in its ability to silence and return gene expression.
Traditionally, CRISPR-Cas9 turned off genes through “knocking it out” of the genome. Crucially, the Cas9 endonuclease protein possesses the ability to cut DNA at sites specified by a guide-RNA complimentary to a nearby DNA sequence. This system originally evolved in bacteria, cutting up the genetic material of invading viruses; however, these cuts marked an irreversible change to the genome. The only way to turn off a gene’s expression was to break it, resulting in defunct proteins with no option to turn the gene back on.
In addition to the irreversible nature of Cas9 edits, the enzyme itself could only cut the DNA, relying on “in-house” repair mechanisms to modify the genome. This method creates inherent unpredictability in the results of a Cas9 cut. “As beautiful as CRISPR-Cas9 is, it hands off the repair to natural cellular processes, which are complex and multifaceted,” says Weissman. “It’s very hard to control the outcomes.”
Rather than make changes to the underlying DNA sequence, Weissman’s research seeks to silence genes at the epigenetic level. DNA in the nucleus is bundled into strands of chromatin. These strands exist as euchromatin, which is loosely bound and can be accessed by the transcription machinery, and heterochromatin, which is tightly bound and cannot be transcribed into RNA. Methylation is a key process in epigenetic modification, often turning euchromatin into transcriptionally silent heterochromatin. CRISPRoff works by methylating DNA at a specific site, meaning the DNA sequence is unchanged, but the newly formed heterochromatin is unable to express proteins. To turn silenced genes back on, the scientists simply used a set of enzymes that demethylate the DNA—a technique dubbed CRISPRon.
The project was partially funded by a 2017 grant from the Defense Advanced Research Projects Agency to create a reversible gene editor. “Fast forward four years [from the initial grant], and CRISPRoff finally works as envisioned in a science fiction way,” says co-senior author Gilbert. “It’s exciting to see it work so well in practice.”
“The big story here is we now have a simple tool that can silence the vast majority of genes,” says Weissman, who is also a professor of biology at MIT and an investigator with the Howard Hughes Medical Institute.“We can do this for multiple genes at the same time without any DNA damage, with great deal of homogeneity, and in a way that can be reversed. It’s a great tool for controlling gene expression.” And while delivery to specific tissues remains a challenge for gene editing technologies such as CRISPRoff, “we showed that you can deliver it transiently as a DNA or as an RNA, the same technology that’s the basis of the Moderna and BioNTech coronavirus vaccine,” Weissman states.
Weissman and collaborators had previously created two other epigenetic editors called CRISPRi and CRISPRa; however, these methods come with the condition that cells had to be continually expressing artificial proteins to maintain the changes. “… current programmable epigenome editing technologies typically rely on constitutive expression of Cas9-fusion proteins to maintain transcriptional control,” the team noted. “As such, these modalities remain less suitable for therapeutic cell and organismal engineering.”
CRISPRoff represents a one-and-done gene editor with the same benefits as CRISPRi and CRISPRa. “With this new CRISPRoff technology, you can [express a protein briefly] to write a program that’s remembered and carried out indefinitely by the cell,” said Gilbert.
The results delivered by CRISPRoff turned out even better than expected, as throughout the course of their research, the scientists made new discoveries about their technology, and about the epigenetic landscape.
While conducting experiments, the researchers discovered that CRISPRoff could be applied to the vast majority of genes, as well as stretches of DNA that control gene expression without coding for an end protein, existing only as DNA or transcribed into RNA’s that don’t function as protein blueprints. “Our initial experiments demonstrate CRISPRoff can perturb enhancers, opening the potential to target genome elements that control tissue-specific gene expression” says first author Nuñez. “That was a huge shock even for us, because we thought it was only going to be applicable for a subset of genes.”
Another surprise came when researchers discovered that epigenetic silencing could occur without the presence of nearby CpG islands (CGIs), which had been thought necessary for DNA methylation. “What was thought before this work was that the 30% of genes that do not have a CpG island were not controlled by DNA methylation,” Gilbert explained. “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.”
A key advantage of CRISPRoff’s epigenetic editing is its heritability. To investigate the viability of CRISPRoff for practical applications, the scientists used induced pluripotent stem (iPS) cells, which hold the potential to differentiate into a wide range of human cell types. After silencing a specific gene in iPS cells, the team of researchers induced the cells to differentiate into neurons. The gene was still turned off in 90% of the resultant neurons, showing that cells modified with CRISPRoff maintained an epigenetic memory, even as they differentiated. “It changes the game so now you’re basically writing a change that is passed down through cell divisions—in some ways we can learn to create a version 2.0 of CRISPR-Cas9 that is safer and just as effective, and can do all these other things as well.”
To further test the therapeutic potential of CRISPRoff, the team targeted the gene coding for Tau protein, which is implicated in Alzheimer’s disease, and found that while Tau protein wasn’t completely silenced, it’s expression was significantly lowered. “What we showed is that this is a viable strategy for silencing Tau and preventing that protein from being expressed,” Weissman commented. “The question is, then, how do you deliver this to an adult? And would it really be enough to impact Alzheimer’s? Those are big open questions, especially the latter.”
In addition to medical applications, the team behind CRISPRoff believes the technology can be used to make more insights into our genome.“Since we now can sort of silence any part of the genome that we want, it’s a great tool for exploring the function of the genome” The authors state. “More generally, this system allows us to broadly explore the biological rules underlying epigenetic silencing and provides a robust tool for controlling gene expression, targeting enhancers, and exploring the principles of epigenetic inheritance.”
Nuñez further stated “I think our tool really allows us to begin to study the mechanism of heritability, especially epigenetic heritability, which is a huge question in the biomedical sciences.”
Original story: An on-off switch for gene editing
Original paper: Nuñez JK, Chen J, Pommier GC, et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell. 2021;0(0). doi: 10.1016/j.cell.2021.03.025
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