This photograph shows Ooceraea biroi workers tagged with color dots for individual behavioral tracking. Credit: Daniel Kronauer The Rockefeller University

The gene-editing technology called CRISPR has revolutionized the way that the function of genes is studied. So far, CRISPR has been widely used to precisely modify single-celled organisms and, more importantly, specific types of cells within more complex organisms. Now, two independent teams of investigators are reporting that CRISPR has been used to manipulate ant eggsleading to germline changes that occur in every cell of the adult animals throughout the entire ant colony. The papers appear August 10 in Cell.

"These studies are proof of principle that you can do genetics in ants," says Daniel Kronauer, an assistant professor at The Rockefeller University and senior author of one of the studies. "If you're interested in studying social behaviors and their genetic basis, ants are a good system. Now, we can knock out any gene that we think will influence social behavior and see its effects."

Because they live in colonies that function like superorganisms, ants are also a valuable model for studying complex biological systems. But ant colonies have been difficult to grow and study in the lab because of the complexity of their life cycles.

The teams found a way to work around that, using two different species of ants. The Rockefeller team employed a species called clonal raider ants (Ooceraea biroi), which lacks queens in their colonies. Instead, single unfertilized eggs develop as clones, creating large numbers of ants that are genetically identical through parthogenesis. "This means that by using CRISPR to modify single eggs, we can quickly grow up colonies containing the gene mutation we want to study," Kronauer says.

The other team, a collaboration between researchers at New York University and the NYU School of Medicine, Arizona State University, the Perelman School of Medicine at the University of Pennsylvania, and Vanderbilt University. , used Indian jumping ants (Harpegnathos saltator). "We chose this species because they have a peculiar feature that makes it easy to transform workers into queens," says Claude Desplan, a Silver Professor at NYU and one of the senior authors of the second study. If the queen dies, the young worker ants will begin dueling for dominance. Eventually, one of them becomes a "pseudoqueen"also called a gamergateand is allowed to lay eggs.

"In the lab, we can inject any worker embryo to change its genetic makeup," Desplan says. "We then convert the worker to a pseudoqueen, which can lay eggs, propagate the new genes, and spawn a new colony."

Desplan, co-senior author Danny Reinberg, a Howard Hughes Medical Institute investigator at NYU Langone, and Shelley Berger, the Daniel S. Och University Professor in the departments of Cell and Developmental Biology and Biology at Penn, began studying these ants several years ago as a way to learn about epigenetics, which refers to changes in gene expression rather than changes in the genetic code itself. "The queens and the worker ants are genetically identical, essentially twin sisters, but they develop very differently," Desplan says. "That makes them a good system for studying epigenetic control of development."

The gene that both research teams knocked out with CRISPR is called orco (odorant receptor coreceptor). Ants have 350 genes for odorant receptors, a prohibitively large number to manage individually. But due to the unique biology of how the receptors worka great stroke of luck, in this casethe investigators were able to block the function of all 350 with a single knockout. "Every one of these receptors needs to team up with the Orco coreceptor in order to be effective," says Waring Trible, a student in Kronauer's lab and the first author of the Rockefeller study.Once the gene was knocked out, the ants were effectively blind to the pheromone signals they normally use to communicate. Without those chemical cues, they become asocial, wandering out of the nest and failing to hunt for food.

More surprisingly, knocking out orco also affected the brain anatomy in the adult animals of both species. In the same way that humans have specialized processing centers in the brain for things like language and facial recognition, ants have centers that are responsible for perceiving and processing olfactory cues that are expanded compared to other insects. But in these ants, the substructures of these sensory centers, called the antennal lobe glomeruli, were largely missing.

"There are many things we still don't know about why this is the case," Kronauer says. "We don't know if the neurons die back in the adults because they're not being used, or if they never develop in the first place. This is something we need to follow up on. And eventually, we'd like to learn to what extent the phenomenon in ants is similar to what's going on in mammals, where brain development does depend to a large extent on sensory input."

"Better understanding, biochemically speaking, how behavior is shaped could reveal insights into disorders in which changes in social communication are a hallmark, such as schizophrenia or depression," Berger says.

In a third related study from the University of Pennsylvania, researchers led by Roberto Bonasio altered ant behavior usingthe brain chemical corazonin. When corazonin is injected into ants transitioning to become a pseudo-queen, it suppresses expression of thebrain protein vitellogenin. This change stimulated worker-like hunting behaviors, while inhibiting pseudo-queen behaviors, such as dueling and egg deposition.

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Further, when the team analyzed proteins the ant brain makes during the transition to becoming a pseudo-queen, they found that corazonin is similar to a reproductive hormone in vertebrates. More importantly, they also discovered that release of corazonin gets turned off as workers became pseudo-queens. Corazonin is also preferentially expressed in workers and foragers from other social insect species. In addition to corazonin, several other genes were expressed in a worker-specific or queen-specific way.

"Social insects such as ants are outstanding models to study how gene regulation affects behavior," says Bonasia, an assistant professor of Cell and Developmental Biology. "This is because they live in colonies comprised of individuals with the same genomes but vastly different sets of behaviors."

Explore further: 'Princess pheromone' tells ants which larvae are destined to be queens

More information: 1. Cell, Trible et al: "orco mutagenesis causes loss of antennal lobe glomeruli and impaired social behavior in ants." http://www.cell.com/cell/fulltext/S0092-8674(17)30772-9 , DOI: 10.1016/j.cell.2017.07.001

2. Cell, Yan et al: "An engineered orco mutation produces aberrant social behavior and defective neural development in ants" http://www.cell.com/cell/fulltext/S0092-8674(17)30770-5 , DOI: 10.1016/j.cell.2017.06.051

3. Cell, Gospocic et al.: "The neuropeptide corazonin controls social behavior and caste identity in ants" http://www.cell.com/cell/fulltext/S0092-8674(17)30821-8 , DOI: 10.1016/j.cell.2017.07.014

Journal reference: Cell

Provided by: Cell Press

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Researchers use CRISPR to manipulate social behavior in ants - Phys.Org

Muscle cells from a patient with myotonic dystrophy type I, untreated (left) and treated with the RNA-targeting Cas9 system (right). The MBNL1 protein is in green, repetitive RNA in red and the cell's nucleus in blue. MBNL1 is an important RNA-binding protein and its normal function is disrupted when it binds repetitive RNA. In the treated cells on the right, MBNL1 is released from the repetitive RNA. Credit: UC San Diego Health

Until recently, the CRISPR-Cas9 gene editing technique could only be used to manipulate DNA. In a 2016 study, University of California San Diego School of Medicine researchers repurposed the technique to track RNA in live cells in a method called RNA-targeting Cas9 (RCas9). In a new study, published August 10 in Cell, the team takes RCas9 a step further: they use the technique to correct molecular mistakes that lead to microsatellite repeat expansion diseases, which include myotonic dystrophy types 1 and 2, the most common form of hereditary ALS, and Huntington's disease.

"This is exciting because we're not only targeting the root cause of diseases for which there are no current therapies to delay progression, but we've re-engineered the CRISPR-Cas9 system in a way that's feasible to deliver it to specific tissues via a viral vector," said senior author Gene Yeo, PhD, professor of cellular and molecular medicine at UC San Diego School of Medicine.

While DNA is like the architect's blueprint for a cell, RNA is the engineer's interpretation of the blueprint. In the central dogma of life, genes encoded in DNA in the nucleus are transcribed into RNA and RNAs carry the message out into the cytoplasm, where they are translated to make proteins.

Microsatellite repeat expansion diseases arise because there are errant repeats in RNA sequences that are toxic to the cell, in part because they prevent production of crucial proteins. These repetitive RNAs accumulate in the nucleus or cytoplasm of cells, forming dense knots, called foci.

In this proof-of-concept study, Yeo's team used RCas9 to eliminate the problem-causing RNAs associated with microsatellite repeat expansion diseases in patient-derived cells and cellular models of the diseases in the laboratory.

Normally, CRISPR-Cas9 works like this: researchers design a "guide" RNA to match the sequence of a specific target gene. The RNA directs the Cas9 enzyme to the desired spot in the genome, where it cuts DNA. The cell repairs the DNA break imprecisely, thus inactivating the gene, or researchers replace the section adjacent to the cut with a corrected version of the gene. RCas9 works similarly but the guide RNA directs Cas9 to an RNA molecule instead of DNA.

The researchers tested the new RCas9 system on microsatellite repeat expansion disease RNAs in the laboratory. RCas9 eliminated 95 percent or more of the RNA foci linked to myotonic dystrophy type 1 and type 2, one type of ALS and Huntington's disease. The approach also eliminated 95 percent of the aberrant repeat RNAs in myotonic dystrophy patient cells cultured in the laboratory.

Another measure of success centered on MBNL1, a protein that normally binds RNA, but is sequestered away from hundreds of its natural RNA targets by the RNA foci in myotonic dystrophy type 1. When the researchers applied RCas9, they reversed 93 percent of these dysfunctional RNA targets in patient muscle cells, and the cells ultimately resembled healthy control cells.

While this study provides the initial evidence that the approach works in the laboratory, there is a long way to go before RCas9 could be tested in patients, Yeo explained.

One bottleneck is efficient delivery of RCas9 to patient cells. Non-infectious adeno-associated viruses are commonly used in gene therapy, but they are too small to hold Cas9 to target DNA. Yeo's team made a smaller version of Cas9 by deleting regions of the protein that were necessary for DNA cleavage, but dispensable for binding RNA.

"The main thing we don't know yet is whether or not the viral vectors that deliver RCas9 to cells would illicit an immune response," he said. "Before this could be tested in humans, we would need to test it in animal models, determine potential toxicities and evaluate long-term exposure."

To do this, Yeo and colleagues launched a spin-out company called Locana to handle the preclinical steps required for moving RCas9 from the lab to the clinic for RNA-based diseases, such as those that arise from microsatellite repeat expansions.

"We are really excited about this work because we not only defined a new potential therapeutic mechanism for CRISPR-Cas9, we demonstrated how it could be used to treat an entire class of conditions for which there are no successful treatment options," said David Nelles, PhD, co-first author of the study with Ranjan Batra, PhD, both postdoctoral researchers in Yeo's lab.

"There are more than 20 genetic diseases caused by microsatellite expansions in different places in the genome," Batra said. "Our ability to program the RCas9 system to target different repeats, combined with low risk of off-target effects, is its major strength."

Explore further: For first time, scientists use CRISPR-Cas9 to target RNA in live cells

More information: Cell (2017). DOI: 10.1016/j.cell.2017.07.010

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New version of DNA editing system corrects underlying defects in RNA-based diseases - Phys.Org

The idea of solving the human organ shortage with pigs has tantalized surgeons for decades. More than 117,000 Americans are currently on a transplant wait-list in the U.S., according to federal figures, and 22 people die every day awaiting a match.

Pig organs are similar in size and function to our own, and people are less squeamish about harvesting body parts from an animal raised for meat than they would be about a primates. Yet one major hurdle that has continued to vex any such cross-species transplants, or xenotransplants, has been the threat of transmitting viruses that can infect people and pigs alike: The latters genome includes 25 so-called retroviruses that apparently do nothing to porkers but might transmit diseases to peopleespecially immune-compromised transplant patients.

That concern, particularly amid the HIV epidemic, has helped stall such research for the past couple decades (with the exception of pig heart valves that are used in humansdead tissue that doesnt pose the same transmission risks). Recent gene editing advances, however, are rejuvenating interest in pig-to-human transplants.

Today scientists in Massachusetts announced that by using the CRISPRCas9 gene-editing system they were able to inactivate all 25 viruses in the pig genome, yielding seemingly healthy piglets and moving research one step closer to a future of xenotransplantation. Our animal is probably the most [genetically] modified animal on the Earth, says Luhan Yang, co-founder and chief science officer of eGenesis, the Cambridge, Mass.based start-up that led the research. We are pushing the envelope of technology day by day. I think that such innovation is required to tackle as challenging a problem as xenotransplantation.

At four months oldroughly the age the pig would need to be for its organs to be large enough to use in peoplethe animals seem perfectly normal, says George Church, a Harvard Medical School geneticist who co-founded eGenesis and is a co-author of the paper. Its a very, very nice piece of work, says Joseph Tector, a transplant surgeon and professor of surgery at the University of Alabama School of Medicine, who was not involved in the research, published in todays Science.

Church says he was surprised the piglets turned out to be so healthy. CRISPR can be toxic to cells because it causes breaks in DNA strands, which can lead cells to self-destruct. Whats more, retroviruses replicate by inserting a copy of their genome into their hosts so those viruses may have been part of the pig genome for the roughly 25 million years that pig species have existed. As a result, Church had wondered if they play an essential role in the pigs survival and whether the animals could develop properly without them.

Another pleasant surprise, Church adds, is that the piglets did not get reinfected with the viruses in the womb. I generally hesitate to say weve solved a two-decade-old problem, but in this case, we have, he notes. So far the team has only made female pigs, raised in a lab. They are now repeating the process to engineer male pigs, which Church says he doesnt expect to be any more complicated.

The next stage of the research, Church and Yang say, will be to essentially humanize the pigsmodifying them enough that their organs can function in the human body. This involves immunological changes as well as making the tissues compatible and fixing blood-clotting issues. They have already begun such work and are writing it up for submission to a peer-reviewed journal, Church adds.

Other teams, including Tectors at Alabama, are working along a similar path, hoping to get the pig parts ready to be tested in the first people within the next two to three years. Researchers expect to start by transplanting kidneys, where the human waiting list is the longest, followed by other organs like the heart and liver; pancreatic islet cells to combat type 1 diabetes; skin; and corneas.

Studying the eGenesis-edited pigs will also give researchers the opportunity to see whether editing a significant number of genes with CRISPR causes any long-term problems in mammals. Pigs are the biggest animals that have undergone CRISPR, he says, and he wants to see what happens when they are allowed to grow to a ripe old age of over 20. There has been some speculation that CRISPR might lead to cancers, but that has not been adequately tested, he says.

Whether or not pig retroviruses would truly pose a risk of causing disease in humans remains controversial. In their new work the Yang team performed experiments confirming that pig retroviruses can infect human cellsjust as another retrovirus, HIV, does with people. In a lab dish the pig viruses infected human cells, and those infected cells were able to infect other human cells that had not been directly exposed to pig cells.

But other researchers say the risk of infecting humans with pig retroviruses is not that clear and that, on balance, unnecessarily editing the pig genes would add to the complexity and cost of a xenotransplant. Tector says his own team stopped worrying about the viruses years ago, because it is not clear whether the U.S. Food and Drug Administration will require the viruses to be removed prior to transplantation. And eGenesiss lab tests did not prove the viruses would be a risk to patients, says Muhammad Mansoor Mohiuddin, a professor of surgery and director of Xenoheart Transplantation at the University of Maryland School of Medicine, who was not part of the new study. The viruses ability to infect cell lines is not enough to be of concern, he says. I fail to understand the significance of this [infectivity] unless it is shown that it can cause some kind of disease.

Still, Tector says, if the FDA does require the viruses to be removed, then the eGenesis teams approach will be useful. If you need to knock [these viruses] out, this is the way to do it, no question, he says.

The rest is here:
Gene-Editing Success Brings Pig-to-Human Transplants Closer to Reality - Scientific American