A new, reversible CRISPR method can control gene expression while keeping the underlying DNA sequence unchanged

Over the past decade, the CRISPR-Cas9 gene editing system has revolutionized genetic engineering, enabling scientists to make targeted changes to the organisms’ DNA. Although the system may be useful for the treatment of various diseases, editing CRISPR-Cas9 involves the cutting of DNA, which results in permanent changes to the genetic material of the cell.

Now, in an article posted online in Cell on April 9, researchers describe a new gene-editing technology called CRISPRoff that allows researchers to control gene expression with high specificity while leaving the sequence of DNA unchanged. The method was designed by Whitehead Institute member Jonathan Weissman, assistant professor at the University of California, Luke Gilbert, postdoctoral fellow James Weñman and Weissman’s associates. The method is stable enough to be inherited by hundreds of cell divisions, and is also completely reversible.

“The big story here is that 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 a researcher at the Howard Hughes Medical Institute. “We can do this for several genes at the same time without any DNA damage, with a lot of homogeneity, and in a way that can be reversed. It’s an excellent tool to control gene expression.”

The project was funded in part by a 2017 grant from the Defense Advanced Research Projects Agency to create a reversible gene editor. “Four years ahead [from the initial grant], and CRISPRoff ultimately works as suggested in a science fiction way, “says fellow senior author Gilbert. It is exciting to see that it works so well in practice. “

Genetic Engineering 2.0

The classic CRISPR-Cas9 system uses a DNA-cutting protein called Cas9 that is found in bacterial immune systems. The system can target specific genes in human cells using a single guide RNA, where the Cas9 proteins cause small breaks in the DNA strand. Then the cell’s existing repair machinery repairs the holes.

Because these methods change the underlying DNA sequence, it is permanent. Moreover, they rely on ‘internal’ mechanisms for cellular repair, that it is difficult to limit the outcome to a single desired change. “As beautiful as CRISPR-Cas9 is, it restores natural cellular processes, which are complex and versatile,” says Weissman. “It is very difficult to control the outcomes.”

This is where the researchers see an opportunity for a different kind of gene editor – one that did not change the DNA sequences themselves, but changed the way they were read in the cell.

This kind of change is what scientists call ‘epigenetic’ – genes can be silenced or activated due to chemical changes to the DNA strand. Problems with the epigenetics of a cell are responsible for many human diseases such as Fragile X syndrome and various cancers, and can be passed down through generations.

Epigenetic gene silence often works by methylation – the addition of chemical labels to certain sites in the DNA strand – that causes the DNA to become inaccessible to RNA polymerase, the enzyme that reads the genetic information in the DNA sequence in messenger- RNA transcripts. may eventually be the blueprints for proteins.

Weissman and associates previously created two other epigenetic editors named CRISPRi and CRISPRa – but both had a caveat. In order for the cells to work, the cells had to constantly express artificial proteins to maintain the changes.

“With this new CRISPRoff technology you can [express a protein briefly] to write a program that is remembered and executed indefinitely by the cell, “says Gilbert. It changes the game, so now you’re basically writing a change that is passed on by cell sections – in some ways we can learn to create a version. 2.0 from CRISPR-Cas9 which is safer and just as effective and can do all these other things as well. “

Build the switch

To build an epigenetic editor that can mimic natural DNA methylation, the researchers created a small protein machine that, led by small RNAs, can stick methyl groups to specific spots on the beach. These methylated genes are then “silenced” or eliminated, hence the name CRISPRoff.

Because the method does not alter the sequence of the DNA strand, the researchers can reverse the silencing effect using enzymes that remove methyl groups, a method they call CRISPRon.

While testing CRISPRoff in different conditions, the researchers discovered some interesting features of the new system. First, they can target the method to the vast majority of genes in the human genome – and this works not only for the genes themselves, but also for other regions of DNA that control gene expression but do not encode proteins. “It was even a huge shock to us because we thought it would only apply to a subset of genes,” says first author Nuñez.

Surprisingly for the researchers, CRISPRoff was also able to silence genes that did not have large methylated regions called CpG islands, which were previously considered necessary for any DNA methylation mechanism.

“What was thought before this work was that the 30 percent of the genes that do not have a CpG island were not controlled by DNA methylation,” says Gilbert. “But our work clearly shows that you do not need a CpG island to eliminate genes by methylation. That was a big surprise to me.”

CRISPRoff in research and therapy

To investigate the potential of CRISPRoff for practical applications, the scientists tested the method in induced pluripotent stem cells. These are cells that can change into innumerable cell types in the body, depending on the molecule to which they are exposed, and are therefore powerful models for studying the development and function of particular cell types.

The researchers chose a gene to silence the stem cells and induced them to become nerve cells called neurons. When searching for the same genes in the neurons, they discovered that it remained silent in 90 percent of the cells, and revealed that the cells retain a memory of epigenetic adaptations made by the CRISPRoff system, even even if it changes the type of cell.

They also selected one gene to use as an example of how CRISPRoff can be applied to therapeutic agents: the gene encoding Tau proteins, which is implicated in Alzheimer’s disease. After testing the method in neurons, they were able to show that the use of CRISPRoff could be used to turn off the Tau expression, although it is not completely off. “What we’ve shown is that it’s a viable strategy to silence Tau and prevent protein from being expressed,” says Weissman. “So the question is how can you deliver it to an adult. And would that really be enough to affect Alzheimer’s? These are big open questions, especially the latter.”

Even if CRISPRoff does not lead to Alzheimer’s therapies, there are many other conditions to which it could potentially be applied. And while delivery to specific tissues remains a challenge for gene editing technologies such as CRISPRoff, “we have shown that you can transmit them briefly as a DNA or as an RNA, the same technology that underlies Modern and BioNTech. coronavirus vaccine, “Weissman says.

Weissman, Gilbert and collaborators are enthusiastic about the potential of CRISPRoff for research as well. “Since we can now silence any part of the genome we want, it is an excellent tool for examining the function of the genome,” says Weissman.

In addition, a reliable system for altering the epigenetics of a cell can help researchers learn the mechanisms by which epigenetic modifications are transmitted through cell division. “I think our tool really allows us to investigate the mechanism of heredity, especially epigenetic heredity, which is a big question in the biomedical sciences,” says Nuñez.