Research Area: Genetics

So-called “junk” DNA plays a key role in speciation
Eva Frederick | Whitehead Institute
August 23, 2021

More than 10 percent of our genome is made up of repetitive, seemingly nonsensical stretches of genetic material called satellite DNA that do not code for any proteins. In the past, some scientists have referred to this DNA as “genomic junk.”

Over a series of papers spanning several years, however, Whitehead Institute Member Yukiko Yamashita and colleagues have made the case that satellite DNA is not junk, but instead has an essential role in the cell: it works with cellular proteins to keep all of a cell’s individual chromosomes together in a single nucleus.

Now, in the latest installment of their work, published online July 24 in the journal Molecular Biology and Evolution, Yamashita and former postdoctoral fellow Madhav Jagannathan, currently an assistant professor at ETH Zurich, Switzerland, take these studies a step further, proposing that the system of chromosomal organization made possible by satellite DNA is one reason that organisms from different species cannot produce viable offspring.

“Seven or eight years ago when we decided we wanted to study satellite DNA, we had zero plans to study evolution,” said Yamashita, who is also a professor of biology at the Massachusetts Institute of Technology and an investigator with the Howard Hughes Medical Institute. “This is one very fun part of doing science: when you don’t have a preconceived idea, and you just follow the lead until you bump into something completely unexpected.”

The origin of species: DNA edition 

Researchers have known for years that satellite DNA is highly variable between species. “If you look at the chimpanzee genome and the human genome, the protein coding regions are, like, 98 percent, 99 percent identical,” she says. “But the junk DNA part is very, very different.”

“These are about the most rapidly evolving sequences in the genome, but the prior perspective has been, ‘Well, these are junk sequences, who cares if your junk is different from mine?’” said Jagannathan.

But as they were investigating the importance of satellite DNA for fertility and survival in pure species, Yamashita and Jagannathan had their first hint that these repetitive sequences might play a role in speciation.

When the researchers deleted a protein called Prod that binds to a specific satellite DNA sequence in the fruit fly Drosophila melanogaster, the flies’ chromosomes scattered outside of the nucleus into tiny globs of cellular material called micronuclei, and the flies died. “But we realized at this point that this [piece of] satellite DNA that was bound by the Prod protein was completely missing in the nearest relatives of Drosophila melanogaster,” Jagannathan said. “It completely doesn’t exist. So that’s an interesting little problem.”

If that piece of satellite DNA was essential for survival in one species but missing from another, it could imply that the two species of flies had evolved different satellite DNA sequences for the same role over time.  And since satellite DNA played a role in keeping all the chromosomes together, Yamashita and Jagannathan wondered whether these evolved differences could be one reason different species are reproductively incompatible.

“After we realized the function [of satellite DNA in the cell], the fact that satellite DNA is quite different between species really hit like lightning,” Yamashita said. “All of a sudden, it became a completely different investigation.”

A tale of two fruit fly species

To study how satellite DNA differences might underlie reproductive incompatibility, the researchers decided to focus on two branches of the fruit fly family tree: the classic lab model Drosophila melanogaster, and its closest relative, Drosophila simulans. These two species diverged from each other two to three million years ago.

Researchers can breed a Drosophila melanogaster female to a Drosophila simulans male, “but [the cross] generates very unhappy offspring,” Yamashita said. “Either they’re sterile or they die.”

Yamashita and Jagannathan bred the flies together, then studied the tissues of the offspring to see what was leading these “unhappy” hybrids to drop like flies. Right away they noticed something interesting: “When we looked at those hybrid tissues, it was very clear that their phenotype was exactly the same as if you had disrupted the satellite DNA [-mediated chromosomal organization] of a pure species,” Yamashita said. “The chromosomes were scattered, and not encapsulated in a single nucleus.”

Furthermore, the researchers could create a healthy hybrid fly by mutating certain genes in the parent flies called “hybrid incompatibility genes,” which have been shown to localize to satellite DNA in the cells of pure species.  Via these experiments, the researchers were able to demonstrate how these genes affect chromosomal packaging in hybrids, and pinpoint the cellular phenotypes associated with them for the first time. “I think for me, that is probably the most critical part of this paper,” Jagannathan said.

Taken together, these findings suggest that because satellite DNA mutates relatively frequently, the proteins that bind the satellite DNA and keep chromosomes together must evolve to keep up, leading each species to develop their own “strategy” for working with the satellite DNA. When two organisms with different strategies interbreed, a clash occurs, leading the chromosomes to scatter outside of the nucleus.

In future studies, Yamashita and Jagannathan hope to put their model to the ultimate test: if they can design a protein that can bind the satellite DNA of two different species and hold the chromosomes together, they could theoretically ‘rescue’ a doomed hybrid, allowing it to survive and produce viable offspring.

This feat of bioengineering is likely years off. “Right now it’s just a pure conceptual thing,” Yamashita said. “In doing this tinkering, there’s probably a lot of specifics that will have to be solved.”

For now, the researchers plan to continue investigating the roles of satellite DNA in the cell, armed with their new knowledge of the part it plays in speciation. “To me, the surprising part of this paper is that our hypothesis was correct,” Jagannathan said. “I mean, in retrospect, there are so many ways things could have been inconsistent with what we hypothesized, so it’s kind of amazing that we’ve sort of been able to chart a clear path from start to finish.”

Rewiring cell division to make eggs and sperm
Whitehead Institute
July 30, 2021

To create eggs and sperm, cells must rewire the process of cell division. Mitosis, the common type of cell division that our bodies use to grow everything from organs to fingernails and to replace aging cells, produces two daughter cells with the same number of chromosomes and approximately the same DNA sequence as the original cell. Meiosis, the specialized cell division that makes egg and sperm in two rounds of cell division, creates four granddaughter cells with new variations in their DNA sequence and half as many chromosomes in each cell. Meiosis uses most of the same cellular machinery as mitosis to achieve this very different outcome; only a few key molecular players prompt the rewiring from one type of division to another. One such key player is the protein Meikin, which is found exclusively in cells undergoing meiosis.

New research from Whitehead Institute Member Iain Cheeseman, graduate student Nolan Maier and collaborators Professor Michael Lampson and senior research scientist Jun Ma at the University of Pennsylvania demonstrates how Meikin is elegantly controlled, and sheds light on how the protein acts to serve multiple roles over different stages of meiosis. The findings, which appear in Developmental Cell on July 30, reveal that Meikin is precisely cut in half midway through meiosis. Instead of this destroying the protein, one half of the molecule, known as C-Meikin, goes on to play a critical role as a previously hidden protein actor in meiosis.

“Cells have this fundamental process, mitosis, during which they have to divide chromosomes evenly or it will cause serious problems like cancer, so the system has to be very robust,” Maier says. “What’s incredible is that you can add one or two unique meiotic proteins like Meikin and dramatically change the whole system very quickly.”

Helping chromosomes stick together

During both mitosis and meiosis, sister chromatids — copies of the same chromosome — pair up to form the familiar “X” shape that we recognize as a chromosome. In mitosis, each chromatid—each half of the X — is connected to a sort of cellular fishing line and these lines reel the chromatids to opposite ends of the cell, where the two new cells are formed around them. However, in the first round of division in meiosis, the sister chromatids stick together, and one whole “X” is reeled into each new cell. Meikin helps to achieve this different outcome by ensuring that, while the chromosomes are being unstuck from each other in preparation for being pulled apart, each pair of sister chromatids stays glued together in the right place. Meikin also helps ensure that certain cellular machinery on the sister chromatids is fused so that they will connect to the same line and be reeled together to the same side of the cell.

More specifically, when chromosomes are first paired up, they are glued together by adhesive molecules in three regions: the centromere, or center of the X, where Meikin localizes; the region around the center; and the arms of the X. In the first round of meiosis, Meikin helps to keep the glue in the region around the center intact, so the sister chromatids will stick together. Simultaneously, Meikin helps to prime the center region to be unglued, while a separate process unglues the arms. This ungluing allows the chromosomes to separate and be prepared for later stages of meiosis.

Cheeseman and Maier initially predicted that Meikin’s role ended after meiosis I, the first round of meiotic cell division. In meiosis II, the second round of cell division, the cells being created should end up with only one sister chromatid each, and so the chromatids must not be kept glued together. Maier found that near the end of meiosis I, Meikin is cleaved in two by an enzyme called Separase, the same molecule that cleaves the adhesive molecules gluing together the chromosomes. At first, this cleavage seemed like the end of Meikin and the end of this story.
A hidden role for a hidden proteinHowever, unexpectedly, the researchers found that cells lacking Meikin during the second half of meiosis do not divide properly, prompting them to take another look at what happens to Meikin after it gets cleaved. They found that Separase cleaves Meikin at a specific point — carving it with the precision of a surgeon’s scalpel — to create C-Meikin, a previously unknown protein that turns out to be necessary for meiosis II. C-Meikin has many of the same properties as the intact Meikin molecule, but it is just different enough to take on a different role: helping to make sure that the chromosomes align properly before their final division.

“There’s a lot of protein diversity in cells that you would never see if you don’t go looking for it, if you only look at the DNA or RNA. In this case, Separase is creating a completely different protein variant of Meikin than can function differently in meiosis II,” says Cheeseman, who is also a professor of biology at Massachusetts Institute of Technology. “I’m very excited to see what we might discover about other hidden protein forms in cell division.”

Recombining ideas

Answering the question of Meikin’s role and regulation throughout meiosis required a close collaboration and partnership between Maier and Lampson lab researcher Ma – the Lampson lab being experts on studying meiosis using mouse models. Working with mouse oocytes (immature egg cells), Ma was able to reveal the behaviors and critical contributions of Meikin cleavage in meiotic cells in mice. Both labs credit the close exchange with helping them to get a deeper understanding of how cells rewire for meiosis.

“It was a pleasure working together to understand how some of the specialized meiotic functions that are necessary for making healthy eggs and sperm are controlled,” Lampson says.

Finally, once cells have completed these specialized meiotic divisions, the researchers found that it was critical for oocytes to fully eliminate Meikin. The researchers determined that, after meiosis two, C-Meikin is degraded by another molecule (the anaphase-promoting complex or APC/C)—this time for good. With Meikin gone and the rewiring of cell division reversed, eggs and sperm are ready for mitosis; should they fuse and form an embryo, that is the next cell division they will undergo. The researchers note that the way Meikin is regulated by being broken down — first into C-Meikin and then completely — may help cells to organize their timing during meiosis. Breaking apart a protein is an irreversible step that creates a clear demarcation between before and after in a multi-step process.The researchers hope that by uncovering the intricacies of meiosis, they may shed light on what happens when the creation of eggs and sperm goes wrong, and so perhaps contribute to our understanding of infertility. Cheeseman also hopes that by studying how mitotic processes are rewired for meiosis, his lab can gain new insights into the original wiring of mitosis.

A “tail” of two RNA regulatory systems
Greta Friar | Whitehead Institute
July 12, 2021

In new research, published in eLife on July 2, Bartel, who is also a professor of biology at Massachusetts Institute of Technology and an investigator with the Howard Hughes Medical Institute, and Bartel lab member Kehui Xiang, a CRI Irvington Postoctoral Fellow, have now discovered how cells establish this early gene regulatory regime and what conditions prompt a switch as the embryos mature. The researchers have observed the same regulatory switch in fish, frogs, and flies, and because the switch occurs across the animal kingdom, they would expect to see that the mechanism applies in other species including mammals.

“When I joined the lab, they had discovered that egg cells and early embryos had this different regulatory regime, and I wanted to know why,” Xiang says. “There must be fundamental changes to the cell, or to the molecules in the cell, that define this.”

The difference in how mRNAs are regulated during and after early development has to do with the length of their tails. mRNAs have tails made up of strings of adenines, one of the building blocks of RNA. Tail length varies between mRNAs from different genes and even between mRNAs from the same gene. Usually, the length of this “poly(A)-tail” corresponds to how long an mRNA lasts before getting degraded. An mRNA with a long tail is more stable, and will generally last longer. However, researchers had also observed that in some cases mRNA tail length corresponds to how readily an mRNA is used to make protein. Bartel’s earlier research had helped define when each of these connections occurs: mRNA tail length affects translational efficiency only in immature eggs and early embryos, and in other stages, it affects mRNA stability or lifespan.

In their new research, Xiang and Bartel uncovered three conditions that are required for the mRNA regulatory regime that exists in early development.

A competitive environment

The first condition is that there has to be a limited availability of a protein that binds to mRNA tails called cytoplasmic poly(A)-binding protein (PABPC). PABPC is known to help activate the translation of mRNA into protein. It binds to the mRNA tail and—in embryos—helps to increase translational efficiency; the researchers propose that it may do this by promoting a more favorable structure for translation. When PABPC is in limited supply, as it is in early embryos, then short-tailed mRNAs are less likely to bind any of the protein, as they will be outcompeted by long-tailed mRNAs, explaining the correlation between tail length and translational efficiency. Later in development, PABPC is in such ready supply that all of the mRNAs are able to bind at least one, decreasing the competitive edge of long-tailed mRNAs.

Early durability

However, the researchers observed that reducing the amount of PABPC in adult cells so that it becomes limiting in these cells did not cause mRNAs with longer tails to be translated more efficiently, which showed that other conditions must also contribute to early embryos’ unique regulation. The second condition that Xiang identified is that mRNAs must be relatively stable in spite of their inability to compete for PABPC. In adult cells, RNAs without PABPC bound to their tails are very unstable, and so are likely to degrade. If the same were true in early embryos, then the short-tailed mRNAs would degrade quickly because they are outcompeted for binding PABPC, and so one would again see a link between tail length and stability, rather than between tail length and translational efficiency—short-tailed mRNAs would be eliminated rather than poorly translated. However, the processes that would normally degrade mRNAs without PABPC have not yet started occurring in early embryos, allowing the short-tailed mRNAs to survive.

Big fish in a small pond

Finally, Xiang discovered that in order for tail length and translational efficiency to be linked, PABPC has to be able to affect translational efficiency. He found that in adult cells PABPC does not appear to boost translational efficiency the way it does in embryos. The researchers hypothesize that this is because the process of translating mRNAs in adult cells is already so efficient that the small boost from binding PABPC does not make a significant difference. However, in early embryos PABPC is more of a big fish in a small pond. The cells do not have all of the machinery to maximize translational efficiency, so every bit of improvement, such as the benefit of binding PABPC, makes a noticeable difference.

Together, these three conditions enable early eggs and embryos to regulate their mRNA in a unique fashion that can control how much protein is made from each gene without destroying the limited pool of mRNA available. In the future, the researchers hope to recreate the three conditions in non-embryonic cells to confirm that the conditions Xiang identified are not only necessary but also sufficient to cause the switch in regulatory regimes.

“Knowing which function the poly(A)-tail is performing in a specific cell or scenario—providing mRNA stability or translational efficiency—is really critical for understanding how genes are regulated in the different cells,” Bartel says. “And understanding that is important for answering all kinds of questions about cells, from their functions to what can go wrong with them in diseases.”

Siniša Hrvatin

Education

  • PhD, 2013, Harvard University
  • A.B., 2007, Biochemical Sciences, Harvard University

Research Summary

To survive extreme environments, many animals have evolved the ability to profoundly decrease metabolic rate and body temperature and enter states of dormancy, such as torpor and hibernation. Our laboratory studies the mysteries of how animals and their cells initiate, regulate, and survive these adaptations. Specifically, we focus on investigating: 1) how the brain regulates torpor and hibernation, 2) how cells adapt to these states, and 3) whether inducing these states can slow down tissue damage, disease progression, and even aging. Our long-term goal is to explore potential applications of inducing similar states of “suspended animation” in humans.

Awards

  • Warren Alpert Distinguished Scholar, Warren Albert Foundation, 2019
  • NIH Director’s New Innovator Award, 2022
  • Searle Scholar, 2023
  • Pew Scholar, 2023
  • McKnight Scholar, 2024
Francisco J. Sánchez-Rivera

Education

  • PhD, 2016, Biology, MIT
  • BS, 2008, Microbiology, University of Puerto Rico at Mayagüez

Research Summary

The overarching goal of the Sánchez-Rivera laboratory is to elucidate the cellular and molecular mechanisms by which genetic variation shapes normal physiology and disease, particularly in the context of cancer. To do so, we develop and apply genome engineering technologies, genetically-engineered mouse models (GEMMs), and single cell lineage tracing and omics approaches to obtain comprehensive biological pictures of disease evolution at single cell resolution. By doing so, we hope to produce actionable discoveries that could pave the way for better therapeutic strategies to treat cancer and other diseases.

Awards

  • V Foundation Award, 2022
  • Hanna H. Gray Fellowship, Howard Hughes Medical Institute, 2018-2026
  • GMTEC Postdoctoral Researcher Innovation Grant, Memorial Sloan Kettering Cancer Center, 2020-2022
  • 100 inspiring Hispanic/Latinx scientists in America, Cell Mentor/Cell Press, 2020
The proteins that package DNA to fit inside cells have another role: tuning gene expression
Raleigh McElvery
May 19, 2021

The DNA inside a single human cell is several meters long — yet it must be condensed to fit inside a space one-tenth the diameter of a hair. That’s like stretching a string from Philadelphia, Pennsylvania to Washington, D.C., and then trying to stuff it into a soccer ball. Imagine then organizing all of this information for each of the body’s 3 trillion cells! The DNA is condensed by proteins called histones that create a spool around which the DNA can wrap itself. How tightly the DNA is wound determines whether it is accessible enough for other proteins to bind to and copy into RNA, toggling gene expression levels up or down.

One specialized type of histone, H2A.Z, is ubiquitous and essential among multicellular organisms. But there have been conflicting reports about how it affects gene expression, especially during embryonic development.

Several years ago, Laurie Boyer’s lab at MIT was the first to show that H2A.Z wraps the DNA located around the start sites of most genes, where the molecular machine RNA polymerase II (RNAPII) binds to copy the DNA into RNA. Boyer’s team demonstrated that removing H2A.Z prevented embryonic cells from turning on genes that are important for forming organs and tissues. But scientists still weren’t sure how H2A.Z exerted its effects.

Now, in a recent Nature Structural and Molecular Biology study, a team from the Boyer lab, led by former postdoc Constantine Mylonas, has revealed how H2A.Z regulates the ability of RNAPII to properly transcribe DNA into the messages that specify all cell types in the body. The researchers found that in embryonic stem cells, H2A.Z serves as a “yellow traffic light,” signaling RNAPII to slow the process of transcribing DNA into RNA. Although there are other proteins that also contribute to RNAPII pausing, H2A.Z establishes a second barrier to transcription that allows gene expression to be tuned in response to developmental signals.

“H2A.Z appears to regulate how fast RNAPII begins to transcribe DNA, and this allows the cell time to respond to important cues that ultimately direct a stem cell to become a brain or heart cell, for example,” says Boyer, a professor of biology and biological engineering. “This connection was a critical missing piece of the puzzle, and explains why H2A.Z is essential for development across all multicellular organisms.”

Illustration of molecules
As RNAPII starts to transcribe a gene, it encounters a cluster of eight histones (a “nucleosome”) including H2A.z, which slows its progression — allowing for tuning of gene expression in response to developmental signals.

According to Boyer, H2A.Z’s role in gene expression has been difficult to pin down because previous approaches only provided static snapshots of how proteins interact with DNA days after loss of the histone. Boyer’s team overcame this shortcoming by leveraging a system that allowed for targeted degradation of H2A.Z within hours. They combined this technique with high-resolution genomic approaches and live cell imaging of RNAPII dynamics using super-resolution microscopy. With help from Ibrahim Cissé’s lab, they were able to visualize RNAPII dynamics in real time at the single molecule level in embryonic stem cells. Upon loss of H2A.Z, they found a remarkable increase in RNAPII movement in the cells, consistent with their genomic results showing a faster release of RNAPII and an increase in transcription in the absence of H2A.Z.

Next, the researchers plan to determine precisely how H2A.Z is targeted to the start sites of genes and how it forms a barrier to RNAPII passage.

Boyer says pinpointing the way histone variants like H2A.Z control gene expression is fundamental to understanding how developmental decisions are made, and will help researchers understand why misregulation of H2A.Z has been linked to diseases such as cancer.

“Emerging evidence indicates that DNA ‘packaging proteins’ like histones directly participate in how RNAPII can read and transcribe DNA,” she explains, “and that crucial connection wasn’t clear before.”

Image credits: courtesy of Laurie Boyer
Top image: Live cell super-resolution imaging showing RNAPII dynamics at a single molecule level in embryonic stem cells. The bright and colored clusters represent RNAPII molecules.

Citation:
“A dual role for H2A. Z. 1 in modulating the dynamics of RNA Polymerase II initiation and elongation.”
Nature Structural & Molecular Biology, online May 10, 2021, DOI: 10.1038/s41594-021-00589-3
Constantine Mylonas, Choongman Lee, Alexander L. Auld, Ibrahim I. Cisse, and Laurie A. Boyer.

Kristin Knouse

Education

  • PhD, 2017, MIT; MD, 2018, Harvard Medical School
  • Undergraduate: BS, 2010, Biology, Duke University

Research Summary

We aim to understand how tissues sense and respond to damage with the goal of developing novel treatments for diverse human diseases. We focus on the mammalian liver, which has the unique ability to completely regenerate itself, in order to identify the molecular requirements for effective organ repair. To this end, we innovate genetic, molecular, and cellular tools that allow us to investigate and modulate organ injury and regeneration directly within living organisms.

Awards

  • NIH Director’s Early Independence Award, 2018
  • Henry Asbury Christian Award, 2018
Olivia Corradin

Education

  • PhD, 2015, Case Western Reserve University
  • BS, 2010, Biochemistry, Marquette University

Research Summary

Our lab studies genetic and epigenetic variation that contributes to human disease by disrupting gene expression programs. We utilize biological insights into the mechanisms of gene regulation in order to determine the impact of disease-associated variants on cellular function. We aim to identify actionable insights into disease pathogenesis by studying the confluence of genetic and epigenetic risk factors of human diseases, including multiple sclerosis and opioid use disorder.

Awards

  • NIH Director’s Pioneer Award Program Avenir Award, 2017
Using CRISPR as a research tool to develop cancer treatments

KSQ Therapeutics uses technology created at MIT to study the role of every human gene in disease biology.

Zach Winn | MIT News Office
April 23, 2021

CRISPR’s potential to prevent or treat disease is widely recognized. But the gene-editing technology can also be used as a research tool to probe and understand diseases.

That’s the basic insight behind KSQ Therapeutics. The company uses CRISPR to alter genes across millions of cells. By observing the effect of turning on and off individual genes, KSQ can decipher their role in diseases like cancer. The company uses those insights to develop new treatments.

The approach allows KSQ to evaluate the function of every gene in the human genome. It was developed at MIT by co-founder Tim Wang PhD ’17 in the labs of professors Eric Lander and David Sabatini.

“Now we can look at every single gene, which you really couldn’t do before in a human cell system, and therefore there are new aspects of biology and disease to discover, and some of these have clinical value,” says Sabatini, who is also a co-founder.

KSQ’s product pipeline includes small-molecule drugs as well as cell therapies that target genetic vulnerabilities identified from their experiments with cancer and tumor cells. KSQ believes its CRISPR-based methodology gives it a more complete understanding of disease biology than other pharmaceutical companies and thus a better chance of developing effective treatments to cancer and other complex diseases.

A tool for discovery

KSQ’s scientific co-founders had been studying the function of genes for years before advances in CRISPR allowed them to precisely edit genomes about 10 years ago. They immediately recognized CRISPR’s potential to help them understand the role of genes in disease biology.

During his PhD work, Wang and his collaborators developed a way to use CRISPR at scale, knocking out individual genes across millions of cells. By observing the impact of those changes over time, the researchers could tease out the functionality of each gene. If a cell died, they knew the gene they knocked out was essential. In cancer cells, the researchers could add drugs and see if knocking out any of the genes affected drug resistance. More sophisticated screening methods taught the researchers how different genes inhibit or drive tumor growth.

“It’s a tool for discovering human biology at scale that was not possible before CRISPR,” says KSQ co-founder Jonathan Weissman, a professor of biology at MIT and a member of the Whitehead Institute. “You can search for genes or mechanisms that can modulate essentially any disease process.”

Wang credits Sabatini with spearheading the commercialization efforts, speaking with investors, and working with MIT’s Technology Licensing Office. Wang also says MIT’s ecosystem helped him think about bringing the technology out of the lab.

“Being at MIT and in the Cambridge area probably made the leap to commercialization a bit easier than it would have been elsewhere,” Wang says. “A lot of the students are entrepreneurial, there’s that rich tradition, so that helped shape my mindset around commercialization.”

Weissman had developed a complementary, CRISPR-based technology that Wang and Sabatini knew would be useful for KSQ’s discovery platform. Around 2015, as the founders were starting the company, they also brought on co-founder William Hahn, a member of the Broad Institute of MIT and Harvard, a professor at Harvard Medical School, and the chief operating officer of the Dana-Farber Cancer Institute.

Since then, the company has advanced Wang’s method.

“They’re able to scale this to a degree that is not possible in any academic lab, even David’s,” Wang says. “The cell lines I used for my experiments were just what was easy to grow and what was in the lab, whereas KSQ is thinking about what therapies aren’t available in certain cancers and deciding what diseases to go after.”

KSQ’s gene evaluations include tens of millions of cells. The company says the data it collects has been predictive of past successes and failures in cancer drug development. Weissman equates the data to “a roadmap for finding cancer vulnerabilities.”

“Cancers have all these different escape routes,” Weissman says. “This is a way of mapping out those escape routes. If there are too many, it’s not a good target to go after, but if there is a small number, you can now start to develop therapies to block off the escape routes.”

From discovery to impact

KSQ’s lead drug candidate is in preclinical development. It targets a DNA-repair pathway identified using an updated version of Wang’s technique. The drug could treat multiple ovarian cancers as well as a disease called triple-negative breast cancer. KSQ is also currently developing a cell therapy to boost the immune system’s ability to fight tumors.

“I’ve always thought the best biotech companies start with information that other people don’t have,” Sabatini says. “I think biotech companies have to have some discovery to them. That’s enabled KSQ to go in different directions.”

The founders feel KSQ has already validated their approach and stimulated further interest in using CRISPR as a research tool.

“There’s a lot of interest in CRISPR as a therapeutic, and that’s an important aspect,” Weissman says. “But I’d argue equally important both in discovery and in therapeutics will be [using CRISPR] to identify the targets you want to go after to affect disease process. Your ability to engineer genomes or make drugs depends on knowing what genes you want to change.”

An on-off switch for gene editing
Eva Frederick | Whitehead Institute
April 9, 2021

Now, in a paper published 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 the DNA unchanged. Designed by Whitehead Institute Member Jonathan Weissman, University of California San Francisco assistant professor Luke Gilbert, Weissman lab postdoc James Nuñez and collaborators, the method is stable enough to be inherited through hundreds of cell divisions, and is also fully reversible.

“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.”

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.”

Genetic engineering 2.0

The classic CRISPR-Cas9 system uses a DNA-cutting protein called Cas9 found in bacterial immune systems. The system can be targeted to specific genes in human cells using a single guide RNA, where the Cas9 proteins create tiny breaks in the DNA strand. Then the cell’s existing repair machinery patches up the holes.

Because these methods alter the underlying DNA sequence, they are permanent. Plus, their reliance on “in-house” cellular repair mechanisms means it is hard to limit the outcome to a single desired change. “As beautiful as CRISPR-Cas9 is, it hands off the repair to natural cellular processes, which are complex and multifaceted,” Weissman says. “It’s very hard to control the outcomes.”

That’s where the researchers saw an opportunity for a different kind of gene editor — one that didn’t alter the DNA sequences themselves, but changed the way they were read in the cell.

This sort of modification is what scientists call “epigenetic” — genes may be silenced or activated based on chemical changes to the DNA strand. Problems with a cell’s epigenetics are responsible for many human diseases such as Fragile X syndrome and various cancers, and can be passed down through generations.

Epigenetic gene silencing often works through methylation — the addition of chemical tags to to certain places in the DNA strand — which causes the DNA to become inaccessible to RNA polymerase, the enzyme which reads the genetic information in the DNA sequence into messenger RNA transcripts, which can ultimately be the blueprints for proteins.

Weissman and collaborators had previously created two other epigenetic editors called CRISPRi and CRISPRa — but both of these came with a caveat. In order for them to work in cells, the cells had to be continually expressing artificial proteins to maintain the changes.

“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,” says Gilbert. “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.”

Building the switch

To build an epigenetic editor that could mimic natural DNA methylation, the researchers created a tiny protein machine that, guided by small RNAs, can tack methyl groups onto specific spots on the strand. These methylated genes are then “silenced,” or turned off, 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 called CRISPRon.

As they tested CRISPRoff in different conditions, the researchers discovered a few interesting features of the new system. For one thing, they could target the method to the vast majority of genes in the human genome — and it worked not just for the genes themselves, but also for other regions of DNA that control gene expression but do not code for proteins. “That was a huge shock even for us, because we thought it was only going to be applicable for a subset of genes,” says first author Nuñez.

Also, surprisingly to the researchers, CRISPRoff was even able to silence genes that did not have large methylated regions called CpG islands, which had previously been thought necessary to any DNA methylation mechanism.

“What was thought before this work was that the 30 percent of genes that do not have a CpG island were not controlled by DNA methylation,” Gilbert says. “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.”

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 turn into countless cell types in the body depending on the cocktail of molecules they are exposed to, and thus are powerful models for studying the development and function of particular cell types.

The researchers chose a gene to silence in the stem cells, and then induced them to turn into nerve cells called neurons. When they looked for the same gene in the neurons, they discovered that it had remained silenced in 90 percent of the cells, revealing that cells retain a memory of epigenetic modifications made by the CRISPRoff system even as they change cell type.

They also selected one gene to use as an example of how CRISPRoff might be applied to therapeutics: the gene that codes for Tau protein, which is implicated in Alzheimer’s disease. After testing the method in neurons, they were able to show that using CRISPRoff could be used to turn Tau expression down, although not entirely off.  “What we showed is that this is a viable strategy for silencing Tau and preventing that protein from being expressed,” Weissman says. “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.”

Even if CRISPRoff does not lead to Alzheimer’s therapies, there are many other conditions it could potentially be applied to. 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 says.

Weissman, Gilbert, and collaborators are enthusiastic about the potential of CRISPRoff for research as well.  “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,” Weissman says.

Plus, having a reliable system to alter a cell’s epigenetics could help researchers learn the mechanisms by which epigenetic modifications are passed down through cell divisions. “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,” Nuñez says.