Scientists Develop CRISPRoff to Control Gene Expression Using Hereditary Epigenetic Memory

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Scientists led by a team at the Whitehead Institute have developed a new gene-editing technology, called CRISPRoff, which can be used to control gene expression with high specificity, while leaving the DNA sequence unchanged. Using a programmable epigenetic memory write protein to silence gene expression, CRISPRoff is a very specific method that efficiently programs epigenetic memory which is inherited for hundreds of cell divisions. Conceived by Jonathan Weissman, fellow of the Whitehead Institute, PhD, Luke Gilbert, PhD, assistant professor at the University of California at San Francisco, James Nuñez, PhD, postdoctoral fellow of the Weissman laboratory, and his collaborators, the method is also fully reversible.

Technology reports in Cell, the developers of CRISORoff, led by Jonathan Weissman, member of the Whitehead Institute, demonstrated that the epigenetic memory of CRISPRoff persists through the differentiation of human-induced pluripotent stem cells (hiPSC) into neurons. “The big story here is that we now have a simple tool that can silence the vast majority of genes,” said Weissman, who is also a professor of biology at MIT and a researcher at the Howard Hughes Medical Institute. “We can do this for multiple genes at the same time without any DNA damage, with great homogeneity and in a way that can be reversed. It is an excellent tool for controlling gene expression.

Weissman and Co-Developers Luke Gilbert, PhD, Assistant Professor, University of California San Francisco, Weissman Lab Postdoctoral Fellow James Nuñez, PhD, and Collaborators, describe the development of CRISPRoff in an article titled “Programmable Transcriptional Memory at the genome scale by CRISPR-based epigenome editing.

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 is finally working as intended in a sci-fi way, ”said co-lead author Gilbert. “It’s exciting to see that it works so well in practice. “

The classic CRISPR-Cas9 system uses a DNA-cutting protein called Cas9, which is found in bacterial immune systems. Using a single guide RNA, the system can target specific genes in human cells, where Cas9 proteins create tiny breaks in the DNA strand. The existing cell repair machinery then plugs the holes.

Because these methods alter the underlying DNA sequence, they are permanent. In addition, their reliance on “homemade” cellular repair mechanisms means that it is difficult to limit the result to a single desired change. “These technologies have been optimized for targeted changes in the underlying DNA sequence and are therefore ideally suited for repairing or introducing pathogenic mutations,” the authors explained. “However, the reliance on endogenous DNA repair machines presents challenges, as the complexity of these pathways can make it difficult to limit the outcome to a single desired change.” Weissman noted: “As beautiful as CRISPR-Cas9 is, it transfers repair to natural cellular processes, which are complex and multifaceted,” Weissman explains. “It’s very difficult to monitor the results. “

The team realized an opportunity to develop another type of gene editor that didn’t alter the DNA sequences themselves, but instead changed the way they were read in the cell. “An alternative modality for modulating gene function is to rewrite the epigenetic landscape to control gene expression without altering the underlying DNA sequence,” they wrote. This epigenetic strategy is designed to silence or activate genes based on chemical changes in the DNA strand. Epigenetic problems in a cell are responsible for many human diseases such as fragile X syndrome and various cancers, and can be passed down from generation to generation.

Epigenetic silencing of genes can work by methylation, that is, adding chemical labels to certain places on the DNA strand, making the DNA inaccessible to the RNA polymerase enzyme which reads the information. genetic DNA sequence in messenger RNA transcripts, which may ultimately represent blueprints for proteins.

Weissman and colleagues had previously created two other epigenetic editors called CRISPRi and CRISPRa, but both came with a caveat. In order for cells to function, cells had to continually express artificial proteins to maintain the changes. “… current programmable epigenome editing technologies generally rely on the constitutive expression of Cas9 fusion proteins to maintain transcriptional control,” the team noted. “As such, these modalities remain less suited to therapeutic cellular and organic engineering.”

To build an epigenetic editor that could mimic the natural methylation of DNA, the researchers created a tiny protein machine that, guided by small RNAs, can glue methyl groups to specific points on the strand. These methylated genes are then silenced, or turned off, hence the name CRISPRoff. Because the method does not change the sequence of the DNA strand, the researchers can reverse the silencing effect through the use of enzymes that remove methyl groups, a method they called CRISPRon.

“With this new CRISPRoff technology, you can [express a protein briefly] to write a program that is memorized and executed indefinitely by the cell, ”Gilbert said. “It’s a game-changer, so now you’re basically writing a change that’s spread through cell divisions. other things too.

By testing CRISPRoff under different conditions, the researchers found that they could target the method on the vast majority of genes in the human genome, and that it also worked not only for the genes themselves, but for other regions of the human genome. DNA that control gene expression. but don’t code for proteins. “Our first experiments demonstrate that CRISPRoff can disrupt enhancers, opening the possibility of targeting elements of the genome that control the expression of tissue-specific genes.” The first author, Nuñez, admitted: “It was a huge shock, even to us, because we thought it would only apply to a subset of genes. “

Additionally, and again surprisingly, CRISPRoff was able to silence genes that lacked large methylated regions called CpG islets (CGIs), which had previously been deemed necessary for any methylation mechanism. DNA. “What was believed prior to this work is that the 30% of genes that do not have a CpG islet were not controlled by DNA methylation,” commented Gilbert. “But our work clearly shows that you don’t need a CpG island to turn off genes through methylation. This, for me, was a major surprise.

To investigate the feasibility of applying CRISPRoff for practical applications, scientists tested the method in induced pluripotent stem cells, which represent useful models for studying the development and function of particular cell types. The researchers chose a gene to silence in the stem cells, then induced the stem cells to differentiate into neurons. Encouragingly, the gene targeted by CRISPRoff remained silent in 90% of the resulting stem cell-derived neurons, revealing that cells retain a memory of epigenetic changes made by the CRISPRoff system even when they change cell type.

The researchers also selected the gene that codes for the Tau protein – which is involved in Alzheimer’s disease – to use as an example of how CRISPRoff could be applied to therapy. After testing the method in neurons, they were able to show that CRISPRoff could be used to reduce Tau expression, but not completely. “What we’ve shown is that this is a viable strategy to silence Tau and prevent expression of this protein,” Weissman said. “The question then is how to deliver this to an adult?” And would that really be enough to have an impact on Alzheimer’s disease? These are big open questions, especially the last one.

Even though CRISPRoff does not lead to therapies for Alzheimer’s disease, there are many other conditions to which it could potentially be applied, the researchers suggest. And while delivery to specific tissues remains a challenge for gene-editing technologies such as CRISPRoff, as Weissman noted, “we’ve shown that you can deliver it transiently as DNA or as DNA. ‘RNA, the same technology that is the basis of the Moderna coronavirus and BioNTech. vaccine.”

Scientists are also excited about CRISPRoff’s potential for research. “Since we can now sort of silence any part of the genome we want, it’s a great tool for exploring genome function,” Weissman noted. “CRISPRoff’s broad ability to initiate inherited genetic silencing even outside of CGIs extends the canonical model of methylation-based silencing and enables various applications including genome-scale screens, multiplexed cell engineering, silencing. enhancers and the mechanistic exploration of epigenetic inheritance, ”the authors concluded.

The availability of a reliable system for modifying a cell’s epigenetics could further help researchers learn more about the mechanisms by which epigenetic modifications are transmitted through cell divisions. As the authors stated, “More generally, this system allows us to broadly explore the biological rules underlying epigenetic silencing and provides a robust tool to control gene expression, target activators, and explore the principles of. epigenetic inheritance. “

Nuñez added, “I think our tool really allows us to start studying the mechanism of heritability, especially epigenetic heritability, which is a huge question in the biomedical sciences.


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