- Dual CRISPR Gene Editing Strategy Cures Animals of HIV-1 Genetic Engineering & Biotechnology News
- UNMC, Temple research increases chances of eliminating HIV infection University of Nebraska Medical Center
- Dual CRISPR therapy plus long-acting ART eliminates HIV in mice FierceBiotech
- CRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretroviral drug-suppressed virus-infected humanized mice | Proceedings of the National Academy of Sciences pnas.org
- Chances of eliminating HIV infection increased by novel dual gene-editing approach Medical Xpress
- View Full Coverage on Google News
Tag Archives: CRISPR
CRISPR tools found in thousands of viruses could boost gene editing
A systematic sweep of viral genomes has revealed a trove of potential CRISPR-based genome-editing tools.
CRISPR–Cas systems are common in the microbial world of bacteria and archaea, where they often help cells to fend off viruses. But an analysis1 published on 23 November in Cell finds CRISPR–Cas systems in 0.4% of publicly available genome sequences from viruses that can infect these microbes. Researchers think that the viruses use CRISPR–Cas to compete with one another — and potentially also to manipulate gene activity in their host to their advantage.
Some of these viral systems were capable of editing plant and mammalian genomes, and possess features — such as a compact structure and efficient editing — that could make them useful in the laboratory.
“This is a significant step forward in the discovery of the enormous diversity of CRISPR–Cas systems,” says computational biologist Kira Makarova at the US National Center for Biotechnology Information in Bethesda, Maryland. “There is a lot of novelty discovered here.”
DNA-cutting defences
Although best known as a tool used to alter genomes in the laboratory, CRISPR–Cas can function in nature as a rudimentary immune system. About 40% of sampled bacteria and 85% of sampled archaea have CRISPR–Cas systems. Often, these microbes can capture pieces of an invading virus’s genome, and store the sequences in a region of their own genome, called a CRISPR array. CRISPR arrays then serve as templates to generate RNAs that direct CRISPR-associated (Cas) enzymes to cut the corresponding DNA. This can allow microbes carrying the array to slice up the viral genome and potentially stop viral infections.
Trove of CRISPR-like gene-cutting enzymes found in microbes
Viruses sometimes pick up snippets of their hosts’ genomes, and researchers had previously found isolated examples of CRISPR–Cas in viral genomes. If those stolen bits of DNA give the virus a competitive advantage, they could be retained and gradually modified to better serve the viral lifestyle. For example, a virus that infects the bacterium Vibrio cholera uses CRISPR–Cas to slice up and disable DNA in the bacterium that encodes antiviral defences2.
Molecular biologist Jennifer Doudna and microbiologist Jillian Banfield at the University of California, Berkeley, and their colleagues decided to do a more comprehensive search for CRISPR–Cas systems in viruses that infect bacteria and archaea, known as phages. To their surprise, they found about 6,000 of them, including representatives of every known type of CRISPR–Cas system. “Evidence would suggest that these are systems that are useful to phages,” says Doudna.
The team found a wide range of variations on the usual CRISPR–Cas structure, with some systems missing components and others unusually compact. “Even if phage-encoded CRISPR–Cas systems are rare, they are highly diverse and widely distributed,” says Anne Chevallereau, who studies phage ecology and evolution at the French National Centre for Scientific Research in Paris. “Nature is full of surprises.”
Small but efficient
Viral genomes tend to be compact, and some of the viral Cas enzymes were remarkably small. This could offer a particular advantage for genome-editing applications, because smaller enzymes are easier to shuttle into cells. Doudna and her colleagues focused on a particular cluster of small Cas enzymes called Casλ, and found that some of them could be used to edit the genomes of lab-grown cells from thale cress (Arabidopsis thaliana), wheat, as well as human kidney cells.
CRISPR ‘cousin’ put to the test in landmark heart-disease trial
The results suggest that viral Cas enzymes could join a growing collection of gene-editing tools discovered in microbes. Although researchers have uncovered other small Cas enzymes in nature, many of those have so far been relatively inefficient for genome-editing applications, says Doudna. By contrast, some of the viral Casλ enzymes combine both small size and high efficiency.
In the meantime, researchers will continue to search microbes for potential improvements to known CRISPR–Cas systems. Makarova anticipates that scientists will also be looking for CRISPR–Cas systems that have been picked up by plasmids — bits of DNA that can be transferred from microbe to microbe.
“Each year we have thousands of new genomes becoming available, and some of them are from very distinct environments,” she says. “So it’s really going to be interesting.”
New CRISPR cancer treatment tested in humans for first time
This article is an installment of Future Explored, a weekly guide to world-changing technology. You can get stories like this one straight to your inbox every Thursday morning by subscribing here.
Past studies have used the gene-editing technology CRISPR to remove genes from immune system cells to make them better at fighting cancer. Now, PACT Pharma and UCLA have used CRISPR to remove and add genes to these cells to help them recognize a patient’s specific tumor cells.
“It is probably the most complicated therapy ever attempted in the clinic,” study co-author Antoni Ribas told Nature. “We’re trying to make an army out of a patient’s own T cells.”
T cells are our immune systems’ built-in defense against cancer.
Natural cancer killers: Our bodies are made up of trillions of cells. These cells grow and multiply through cell division, and when they get old or become damaged, they die and new cells replace them.
Cancerous cells have genetic mutations that prevent them from dying when they should — instead, they multiply uncontrollably, potentially forming clumps or spreading to other parts of the body and crowding out healthy cells.
Our immune system includes a built-in defense against cancer: T cells. These are a type of white blood cell with proteins on their surfaces, called “T cell receptors,” that bind to specific foreign or cancerous cells, prompting the T cell to attack and kill them.
The challenge: T cells aren’t perfect, though.
In part because cancer cells look a lot like healthy cells, they’re adept at flying under the immune system’s radar. Tumor cells can also release chemical signals that make them even harder for T cells to identify.
“At some point the immune system kind of lost the battle and the tumor grew.”
Stefanie Mandl
Sometimes cancer cells are simply multiplying too fast for T cells to keep up.
“If [T cells] see something that looks not normal, they kill it,” lead author Stefanie Mandl, who serves as CSO of PACT Pharma, told Nature. “But in the patients we see in the clinic with cancer, at some point the immune system kind of lost the battle and the tumor grew.”
By genetically engineering T cells to be better at spotting proteins commonly found on the surfaces of blood cancer cells, researchers have been able to develop treatments — called “CAR-T cell therapies” — for people with those cancers.
No one has yet been able to find reliable, comparable proteins that work for solid tumors, though — each person’s cancer seems to be too unique for existing CAR-T cell therapies to be effective.
CRISPR boost: A small phase 1 study by researchers at PACT Pharma and UCLA suggests that we may be able to use CRISPR to boost the ability of our T cells to fight solid cancers.
They took blood and tumor samples from 16 patients with solid tumors in various parts of their bodies. Using genetic sequencing, they scoured the samples for mutations that were present in their tumor cells but not their blood cells.
“The mutations are different in every cancer,” said Ribas. “And although there are some shared mutations, they are the minority.”
“We’re trying to make an army out of a patient’s own T cells.”
Antoni Ribas
The researchers then searched each participant’s T cells, looking for ones with receptors likely to recognize the cancer’s mutations.
Using CRISPR, they knocked out a gene for an existing receptor and inserted a gene for a cancer-targeting receptor into T cells that lacked it. Once they had engineered what they thought would be enough T cells, the researchers infused them back into the patient.
The results: Later biopsies found that up to 20% of the immune cells in the patients’ tumors were the engineered T cells, suggesting that those cells were in fact very adept at homing in on the cancer.
Only two of the 16 participants experienced minor side effects — fever, chills, confusion — attributable to the T cells, but they quickly resolved.
“It is probably the most complicated therapy ever attempted in the clinic.”
Antoni Ribas
A month after treatment, five of the patients’ tumors were the same size as before, suggesting that the engineered cells may have had a stabilizing effect on their condition.
The cancer continued to progress in the other 11 patients, but the patient given the highest dose of cells saw a short term improvement in their cancer — that could mean the treatment would be more effective in future studies if administered in higher doses.
“We just need to hit it stronger the next time,” said Ribas.
The cold water: This small phase 1 suggests that the engineered T cells are safe and potentially effective, but there are still a lot of limitations to overcome.
One problem is that while the engineered T cells did tend to home in on the tumor, they didn’t always attack the cancer cells.
“The fact that you can get those T cells into a tumor is one thing. But if they get there and don’t do anything, that’s disappointing,” Bruce Levine, a professor of cancer gene therapy at the University of Pennsylvania, who wasn’t involved in the study, told WIRED.
Another is that the treatment is expensive, complicated, and time-consuming — it took the researchers a median of 5.5 months just to identify the mutations to target with CRISPR after sequencing a patient’s cells.
“You can build on this. You can make it better and more potent and faster.”
Katy Rezvani
Looking ahead: These hurdles aren’t insurmountable, and now that the researchers have shown that CRISPR can be used to engineer cancer-targeting T cells, future studies can take the approach to the next level.
“You can build on this,” oncologist Katy Rezvani, who wasn’t involved in the study, told Medical Express. “You can make it better and more potent and faster.”
One day, the engineered T cells might even allow doctors to protect their patients against recurrence while treating their existing cancer.
“We are reprogramming a patient’s immune system to target their own cancer,” Mandl told TIME. “It’s a living drug, so you can give one dose and ideally have life-long protection [from the cancer].”
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Silver bullet for cancer? Scientists use CRISPR to unlock patients’ true tumor-fighting potential
Scientists have tailored DNA-editing technology to turbocharge how the body fights cancer cells — in a potential breakthrough.
They modified patients’ genes to instruct cancer-fighting cells to swarm tumors using CRISPR, which is given as a one-off injection.
CRISPR has been previously used in humans to remove specific genes to allow the immune system to be more activated against cancer.
But the new study was able to not only take out specific genes, but insert new ones which program immune cells to fight the patient’s own specific cancer.
Dr Antoni Ribas, from the University of California, Los Angeles and co-leader of the study said: ‘This is a leap forward in developing a personalized treatment for cancer.’
Scientists at pharmaceutical company PACT Pharma used gene-editing technology to isolate and clone cancer patients’ immune cells and prime them to target mutations on cancer cells.
Researchers took blood and tumor samples from 16 patients with various forms of cancer including including colon, breast and lung.
They isolated the immune cells that had hundreds of mutations targeted specifically at the cancers plaguing their bodies.
These were modified to be able to target each patient’s specific tumor, which have hundreds of unique mutations.
One month after treatment, five of the participants experienced stable disease, meaning their tumors had not grown.
The CRISPR tool consists of two main actors: a guide RNA and a DNA-cutting enzyme. The guide RNA is a specific RNA sequence that recognizes the target piece of DNA to be edited and directs the enzyme, Cas9, to initiate the editing process.
Cas9 precisely cuts the target strands of DNA and removes a small piece, causing a gap in the DNA where a new piece of DNA can be added.
Scientists design the guide RNA to mirror the DNA of the gene to be edited, known as the target.
The guide RNA partners with the Cas9 enzyme and leads it to the target gene. When the guide RNA matches up with the target gene’s DNA, Cas9 splices off the DNA, shutting the targeted gene off.
Since the CRISPR technique has been around for about a decade and remains at the center of ambitious scientific projects.
Doctors are now exploring its application in treating rare diseases and genetic disorders such as sickle cell disease.
‘The generation of a personalized cell treatment for cancer would not have been feasible without the newly developed ability to use the CRISPR technique to replace the immune receptors in clinical-grade cell preparations in a single step,’ Dr Ribas added.
The findings give hope for 1.9 million Americans who will be diagnosed with some form of cancer this year.
Roughly 290,000 women and 2,700 men will be diagnosed with breast cancer, which makes it the most common cancer diagnosis.
Prostate cancer is the leading cancer diagnosis among men and the second most common diagnosis overall with about 269,000 expected cases this year.
Still, the technology is relatively new and poses some hefty ethical questions about its application for genetic remodeling.
Medicine has entered uncharted territory in which hereditary disabilities in an embryo could possibly be edited out.
Safety issues in gene editing technology research are not unheard of.
There is a risk of erroneously changing the DNA or RNA in regions other than the target site, which could result in unwanted side effects not just in the patient but in future generations.
A major scandal rocked the world in 2019 when Chinese scientist He Jiankui was imprisoned after modifying the DNA of twin girls Lulu and Nana before birth to make them resistant to HIV.
He’s work to manipulate the genes of human embryos was deemed ‘monstrous,’ ‘unethical,’ and ‘very dangerous’.
A group of over 100 scientists in China blasted He’s work in 2018: ‘Conducting direct human experiments can only be described as crazy.’
The group added, ‘Pandora’s box has been opened. We still might have a glimmer of hope to close it before it’s too late.’
In 2019, a group of scientists proposed a worldwide moratorium on human germline editing.
They wrote: ‘By ‘global moratorium’, we do not mean a permanent ban. Rather, we call for the establishment of an international framework in which nations, while retaining the right to make their own decisions, voluntarily commit to not approve any use of clinical germline editing unless certain conditions are met.’
PACT Pharma’s findings were published Thursday in the journal Nature.
A bold effort to cure HIV—using Crispr
Enlarge / A 3D illustration of the HIV virus.
In July, an HIV-positive man became the first volunteer in a clinical trial aimed at using Crispr gene editing to snip the AIDS-causing virus out of his cells. For an hour, he was hooked up to an IV bag that pumped the experimental treatment directly into his bloodstream. The one-time infusion is designed to carry the gene-editing tools to the man’s infected cells to clear the virus.
Later this month, the volunteer will stop taking the antiretroviral drugs he’s been on to keep the virus at undetectable levels. Then, investigators will wait 12 weeks to see if the virus rebounds. If not, they’ll consider the experiment a success. “What we’re trying to do is return the cell to a near-normal state,” says Daniel Dornbusch, CEO of Excision BioTherapeutics, the San Francisco-based biotech company that’s running the trial.
The HIV virus attacks immune cells in the body called CD4 cells and hijacks their machinery to make copies of itself. But some HIV-infected cells can go dormant—sometimes for years—and not actively produce new virus copies. These so-called reservoirs are a major barrier to curing HIV.
“HIV is a tough foe to fight because it’s able to insert itself into our own DNA, and it’s also able to become silent and reactivate at different points in a person’s life,” says Jonathan Li, a physician at Brigham and Women’s Hospital and HIV researcher at Harvard University who’s not involved with the Crispr trial. Figuring out how to target these reservoirs—and doing it without harming vital CD4 cells—has proven challenging, Li says.
While antiretroviral drugs can halt viral replication and clear the virus from the blood, they can’t reach these reservoirs, so people have to take medication every day for the rest of their lives. But Excision BioTherapeutics is hoping that Crispr will remove HIV for good.
Crispr is being used in several other studies to treat a handful of conditions that arise from genetic mutations. In those cases, scientists are using Crispr to edit peoples’ own cells. But for the HIV trial, Excision researchers are turning the gene-editing tool against the virus. The Crispr infusion contains gene-editing molecules that target two regions in the HIV genome important for viral replication. The virus can only reproduce if it’s fully intact, so Crispr disrupts that process by cutting out chunks of the genome.
In 2019, researchers at Temple University and the University of Nebraska found that using Crispr to delete those regions eliminated HIV from the genomes of rats and mice. A year later, the Temple group also showed that the approach safely removed viral DNA from macaques with SIV, the monkey version of HIV.
New CRISPR-based map ties every human gene to its function | MIT News
The Human Genome Project was an ambitious initiative to sequence every piece of human DNA. The project drew together collaborators from research institutions around the world, including MIT’s Whitehead Institute for Biomedical Research, and was finally completed in 2003. Now, over two decades later, MIT Professor Jonathan Weissman and colleagues have gone beyond the sequence to present the first comprehensive functional map of genes that are expressed in human cells. The data from this project, published online June 9 in Cell, ties each gene to its job in the cell, and is the culmination of years of collaboration on the single-cell sequencing method Perturb-seq.
The data are available for other scientists to use. “It’s a big resource in the way the human genome is a big resource, in that you can go in and do discovery-based research,” says Weissman, who is also a member of the Whitehead Institute and an investigator with the Howard Hughes Medical Institute. “Rather than defining ahead of time what biology you’re going to be looking at, you have this map of the genotype-phenotype relationships and you can go in and screen the database without having to do any experiments.”
The screen allowed the researchers to delve into diverse biological questions. They used it to explore the cellular effects of genes with unknown functions, to investigate the response of mitochondria to stress, and to screen for genes that cause chromosomes to be lost or gained, a phenotype that has proved difficult to study in the past. “I think this dataset is going to enable all sorts of analyses that we haven’t even thought up yet by people who come from other parts of biology, and suddenly they just have this available to draw on,” says former Weissman Lab postdoc Tom Norman, a co-senior author of the paper.
Pioneering Perturb-seq
The project takes advantage of the Perturb-seq approach that makes it possible to follow the impact of turning on or off genes with unprecedented depth. This method was first published in 2016 by a group of researchers including Weissman and fellow MIT professor Aviv Regev, but could only be used on small sets of genes and at great expense.
The massive Perturb-seq map was made possible by foundational work from Joseph Replogle, an MD-PhD student in Weissman’s lab and co-first author of the present paper. Replogle, in collaboration with Norman, who now leads a lab at Memorial Sloan Kettering Cancer Center; Britt Adamson, an assistant professor in the Department of Molecular Biology at Princeton University; and a group at 10x Genomics, set out to create a new version of Perturb-seq that could be scaled up. The researchers published a proof-of-concept paper in Nature Biotechnology in 2020.
The Perturb-seq method uses CRISPR-Cas9 genome editing to introduce genetic changes into cells, and then uses single-cell RNA sequencing to capture information about the RNAs that are expressed resulting from a given genetic change. Because RNAs control all aspects of how cells behave, this method can help decode the many cellular effects of genetic changes.
Since their initial proof-of-concept paper, Weissman, Regev, and others have used this sequencing method on smaller scales. For example, the researchers used Perturb-seq in 2021 to explore how human and viral genes interact over the course of an infection with HCMV, a common herpesvirus.
In the new study, Replogle and collaborators including Reuben Saunders, a graduate student in Weissman’s lab and co-first author of the paper, scaled up the method to the entire genome. Using human blood cancer cell lines as well noncancerous cells derived from the retina, he performed Perturb-seq across more than 2.5 million cells, and used the data to build a comprehensive map tying genotypes to phenotypes.
Delving into the data
Upon completing the screen, the researchers decided to put their new dataset to use and examine a few biological questions. “The advantage of Perturb-seq is it lets you get a big dataset in an unbiased way,” says Tom Norman. “No one knows entirely what the limits are of what you can get out of that kind of dataset. Now, the question is, what do you actually do with it?”
The first, most obvious application was to look into genes with unknown functions. Because the screen also read out phenotypes of many known genes, the researchers could use the data to compare unknown genes to known ones and look for similar transcriptional outcomes, which could suggest the gene products worked together as part of a larger complex.
The mutation of one gene called C7orf26 in particular stood out. Researchers noticed that genes whose removal led to a similar phenotype were part of a protein complex called Integrator that played a role in creating small nuclear RNAs. The Integrator complex is made up of many smaller subunits — previous studies had suggested 14 individual proteins — and the researchers were able to confirm that C7orf26 made up a 15th component of the complex.
They also discovered that the 15 subunits worked together in smaller modules to perform specific functions within the Integrator complex. “Absent this thousand-foot-high view of the situation, it was not so clear that these different modules were so functionally distinct,” says Saunders.
Another perk of Perturb-seq is that because the assay focuses on single cells, the researchers could use the data to look at more complex phenotypes that become muddied when they are studied together with data from other cells. “We often take all the cells where ‘gene X’ is knocked down and average them together to look at how they changed,” Weissman says. “But sometimes when you knock down a gene, different cells that are losing that same gene behave differently, and that behavior may be missed by the average.”
The researchers found that a subset of genes whose removal led to different outcomes from cell to cell were responsible for chromosome segregation. Their removal was causing cells to lose a chromosome or pick up an extra one, a condition known as aneuploidy. “You couldn’t predict what the transcriptional response to losing this gene was because it depended on the secondary effect of what chromosome you gained or lost,” Weissman says. “We realized we could then turn this around and create this composite phenotype looking for signatures of chromosomes being gained and lost. In this way, we’ve done the first genome-wide screen for factors that are required for the correct segregation of DNA.”
“I think the aneuploidy study is the most interesting application of this data so far,” Norman says. “It captures a phenotype that you can only get using a single-cell readout. You can’t go after it any other way.”
The researchers also used their dataset to study how mitochondria responded to stress. Mitochondria, which evolved from free-living bacteria, carry 13 genes in their genomes. Within the nuclear DNA, around 1,000 genes are somehow related to mitochondrial function. “People have been interested for a long time in how nuclear and mitochondrial DNA are coordinated and regulated in different cellular conditions, especially when a cell is stressed,” Replogle says.
The researchers found that when they perturbed different mitochondria-related genes, the nuclear genome responded similarly to many different genetic changes. However, the mitochondrial genome responses were much more variable.
“There’s still an open question of why mitochondria still have their own DNA,” said Replogle. “A big-picture takeaway from our work is that one benefit of having a separate mitochondrial genome might be having localized or very specific genetic regulation in response to different stressors.”
“If you have one mitochondria that’s broken, and another one that is broken in a different way, those mitochondria could be responding differentially,” Weissman says.
In the future, the researchers hope to use Perturb-seq on different types of cells besides the cancer cell line they started in. They also hope to continue to explore their map of gene functions, and hope others will do the same. “This really is the culmination of many years of work by the authors and other collaborators, and I’m really pleased to see it continue to succeed and expand,” says Norman.
CRISPR Gene Editing Now Possible in Cockroaches
Cartoon of CRISPR in cockroaches. Credit: Shirai et al./Cell Reports Methods
According to a paper published in the journal Cell Reports Methods by Cell Press on May 16th, 2022, researchers devised a CRISPR-Cas9 technique to enable gene editing in cockroaches. The straightforward and effective “direct parental” CRISPR (DIPA-CRISPR) procedure involves injecting materials into female adults where eggs are developing rather than into the embryos themselves.
“In a sense, insect researchers have been freed from the annoyance of egg injections,” says senior study author Takaaki Daimon of Kyoto University. “We can now edit insect genomes more freely and at will. In principle, this method should work for more than 90% of insect species.”
“By improving the DIPA-CRISPR method and making it even more efficient and versatile, we may be able to enable genome editing in almost all of the more than 1.5 million species of insects, opening up a future in which we can fully utilize the amazing biological functions of insects.” — Takaaki Daimon
Current approaches for insect gene editing typically require microinjection of materials into early embryos, severely limiting its application to many species. For example, past studies have not achieved genetic manipulation of cockroaches due to their unique reproductive system. In addition, insect gene editing often requires expensive equipment, a specific experimental setup for each species, and highly skilled laboratory personnel. “These problems with conventional methods have plagued researchers who wish to perform genome editing on a wide variety of insect species,” Daimon says.
To overcome these limitations, Daimon and his collaborators injected Cas9 ribonucleoproteins (RNPs) into the main body cavity of adult female cockroaches to introduce heritable mutations in developing egg cells. The results demonstrated that gene editing efficiency—the proportion of edited individuals out of the total number of individuals hatched—could reach as high as 22%. In the red flour beetle, DIPA-CRISPR achieved an efficiency of more than 50%. Moreover, the researchers generated gene knockin beetles by co-injecting single-stranded oligonucleotides and Cas9 RNPs, but the efficiency is low and should be further improved.
The successful application of DIPA-CRISPR in two evolutionarily distant species demonstrates its potential for broad use. But the approach is not directly applicable to all insect species, including fruit flies. In addition, the experiments showed that the most critical parameter for success is the stage of the adult females injected. As a result, DIPA-CRISPR requires good knowledge of ovary development. This can be challenging in some species, given the diverse life histories and reproductive strategies in insects.
Despite these limitations, DIPA-CRISPR is accessible, highly practical, and could be readily implemented in laboratories, extending the application of gene editing to a wide diversity of model and non-model insect species. The technique requires minimal equipment for adult injection, and only two components—Cas9 protein and single-guide
CRISPR now possible in cockroaches
Cell Reports Methods” width=”800″ height=”530″/>
Cartoon of CRISPR in cockroaches. Credit: Shirai et al./Cell Reports Methods
Researchers have developed a CRISPR-Cas9 approach to enable gene editing in cockroaches, according to a study published by Cell Press on May 16th in the journal Cell Reports Methods. The simple and efficient technique, named “direct parental” CRISPR (DIPA-CRISPR), involves the injection of materials into female adults where eggs are developing rather than into the embryos themselves.
“In a sense, insect researchers have been freed from the annoyance of egg injections,” says senior study author Takaaki Daimon of Kyoto University. “We can now edit insect genomes more freely and at will. In principle, this method should work for more than 90% of insect species.”
Current approaches for insect gene editing typically require microinjection of materials into early embryos, severely limiting its application to many species. For example, past studies have not achieved genetic manipulation of cockroaches due to their unique reproductive system. In addition, insect gene editing often requires expensive equipment, a specific experimental setup for each species, and highly skilled laboratory personnel. “These problems with conventional methods have plagued researchers who wish to perform genome editing on a wide variety of insect species,” Daimon says.
To overcome these limitations, Daimon and his collaborators injected Cas9 ribonucleoproteins (RNPs) into the main body cavity of adult female cockroaches to introduce heritable mutations in developing egg cells. The results demonstrated that gene editing efficiency—the proportion of edited individuals out of the total number of individuals hatched—could reach as high as 22%. In the red flour beetle, DIPA-CRISPR achieved an efficiency of more than 50%. Moreover, the researchers generated gene knockin beetles by co-injecting single-stranded oligonucleotides and Cas9 RNPs, but the efficiency is low and should be further improved.
The successful application of DIPA-CRISPR in two evolutionarily distant species demonstrates its potential for broad use. But the approach is not directly applicable to all insect species, including fruit flies. In addition, the experiments showed that the most critical parameter for success is the stage of the adult females injected. As a result, DIPA-CRISPR requires good knowledge of ovary development. This can be challenging in some species, given the diverse life histories and reproductive strategies in insects.
Despite these limitations, DIPA-CRISPR is accessible, highly practical, and could be readily implemented in laboratories, extending the application of gene editing to a wide diversity of model and non-model insect species. The technique requires minimal equipment for adult injection, and only two components—Cas9 protein and single-guide RNA—greatly simplifying procedures for gene editing. Moreover, commercially available, standard Cas9 can be used for adult injection, eliminating the need for time-consuming custom engineering of the protein.
“By improving the DIPA-CRISPR method and making it even more efficient and versatile, we may be able to enable genome editing in almost all of the more than 1.5 million species of insects, opening up a future in which we can fully utilize the amazing biological functions of insects,” Daimon says. “In principle, it may be also possible that other arthropods could be genome edited using a similar approach. These include agricultural and medical pests such as mites and ticks, and important fishery resources such as shrimp and crabs.”
World’s first gene editing tools for ticks may help decrease tick-borne diseases
More information:
Takaaki Daimon, DIPA-CRISPR is a simple and accessible method for insect gene editing, Cell Reports Methods (2022). DOI: 10.1016/j.crmeth.2022.100215. www.cell.com/cell-reports-meth … 2667-2375(22)00078-9
Takaaki Daimon, DIPA-CRISPR is a simple and accessible method for insect gene editing, Cell Reports Methods (2022). DOI: 10.1016/j.crmeth.2022.100215. www.cell.com/cell-reports-meth … 2667-2375(22)00078-9
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Hypoallergenic Cats Could Be Possible With CRISPR Gene Editing
A team of researchers say they’ve found an effective way to block the most common source of cat allergies using the gene-editing technology CRISPR. Their findings also suggest that hypoallergenic cats can be just as healthy as the typical feline.
Allergies are most associated with the fur and dander that cats shed into the environment, but those aren’t the true culprit. A protein produced by cats called Fel d 1—which ends up in their saliva and tears and, by extension, the fur that they’re constantly cleaning—is thought to cause over 90% of cat allergies. This has made the protein an appealing target for scientists trying to reduce the burden of cat allergies, which may affect up to 20% of people.
Researchers at the Virginia-based biotech company InBio (previously called Indoor Biotechnologies) have been working on their own approach. They’re hoping to use CRISPR, the Nobel Prize-winning gene editing tech, to produce cats that simply make little to no Fel d 1. In their latest research, published Monday in The CRISPR Journal, they say they’ve collected evidence that this can be done effectively and safely.
Analyzing the DNA of 50 domestic cats, they found regions along two genes primarily involved in producing Fel d 1 that would be suitable for editing with CRISPR. When they compared the genes of these cats to those from eight wild cat species, they also found that there was a lot of variation between the groups. That could indicate, as other research has suggested, that Fel d 1 is non-essential to cat biology and can thus be eliminated without any health risks. (Some cat breeds, like the Russian blue and Balinese, are often touted as being better for people with allergies because they may naturally produce less Fel d 1.) Lastly, the team used CRISPR on cat cells in the lab, which seemed to be effective at knocking out Fel d 1 and appeared to produce no off-target edits in the areas they predicted that edits would most likely happen.
All in all, the researchers say that their findings show that “Fel d 1 is both a rational and viable candidate for gene deletion, which may profoundly benefit cat allergy sufferers by removing the major allergen at the source.”
There are plenty of efforts ongoing to create less sneeze-inducing cats. In early 2020, pet food company Purina released its LiveClear line of products—cat food that’s been treated with an egg-based protein that inhibits the Fel d 1 in their mouths. The company’s research has found that levels of Fel d 1 in cat fur and dander drop by an average 47% after three weeks of cats eating LiveClear. Elsewhere, other researchers have been working on a vaccine for cats that trains their immune system to reduce levels of the protein.
The authors of the new study note that modestly reducing the amount of Fel d 1 produced by cats may be possible in lots of ways. But since, as any cat owner knows, cats are constantly shedding fur, it’s still possible for smaller amounts of Fel d 1 to accumulate in house dust and pose a major allergic threat. By targeting Fel d 1 at its root using gene-editing, they argue, it may be possible to create truly hypoallergenic cats.
Of course, this is all still early days. The scientists plan to continue refining and testing out their technique, both in the lab and eventually in real-life cats genetically bred to have their Fel d 1 knocked out. Should that work out as hoped, with no adverse effects, the next step would be to find a way to safely genetically edit adult cats.
30-year-old female founder’s billion-dollar bet on CRISPR gene editing
Janice Chen (C) and her Mammoth Biosciences co-founders Trevor Martin (L) and Lucas Harrington (R). CRISPR gene editing pioneer and Nobel Prize winner Jennifer Doudna is also a co-founder.
Along Highway 101 north of the San Francisco Airport, a break-out biotech start-up named Mammoth Biosciences co-founded by Nathan Chen’s sister Janice in 2018 is fast emerging in the revolutionary field of CRISPR technology.
While not high profile like her gold medal-winning, ice skating brother — or Mammoth co-founder Jennifer Doudna, who won a Nobel Prize in chemistry for her work on CRISPR — Chen’s bioscience work in gene editing technology is in the forefront of medical discoveries from identifying bacterial and viral infections to early cancer detection.
CRISPR, or clustered regularly interspaced short palindromic repeats, effectively cuts genomes and slices DNA to treat genetic diseases.
Outside of a close circle of colleagues, few knew Nathan was her sibling until she excitedly posted on social media about his gold medal victory as her family watched the televised games from her San Francisco home. Chen recalls being with her family in Seoul four years ago and watching him compete in the 2018 Winter Olympics. During breaks, she was busy contacting lawyers to start the process of setting up the company.
Since the pandemic in 2020, the biotech start-up has fast accelerated. The company nabbed approximately $100 million in contracts with Bayer and Vertex Pharmaceuticals and government grants, grew the employee count from 30 to 130, and is hiring at least 55 more. Its valuation soared to $1 billion, with $150 million in a venture deal last September that included Amazon, famed Silicon Valley VC firm Mayfield and Apple’s Tim Cook.
The exit strategy isn’t an acquisition, as Chen sees it.
“Our intention is not to build and sell it but to become a $100 billion company in next-generation CRISPR technology. There are so many creative building opportunities, and new technology that can come out of discovery in gene editing,” said Chen. “Identifying the business strategy has meant that I needed to step out of the lab and scale the company,” added Chen, who worked remotely during Covid, but is now back at the company’s Brisbane, California, headquarters, where its distinct green and white elephant-shaped signage is highly visible.
Salt Lake City roots, Silicon Valley growth
Growing up in Salt Lake City as one of five siblings (Nathan, 22, is the youngest), her parents, immigrants from China in 1988, encouraged “us to reach our potential and become what is best for us,” Chen, now 30, said. Chen learned to play the violin, competed in chess tournaments, and excelled in dance performance. In chess competitions, where she was often the youngest and the only female, she said she learned “how to lose and how to win strategies.”
She discovered her passion for bioscience while at her father’s small biotech business in Utah.
To relieve the stress of scaling up Mammoth Biosciences, Chen has recently taken up running in San Francisco’s hills, near her home. She got up to speed for on-the-job managerial challenges by reading “The Founder’s Dilemma.” She also sought the advice of an executive coach who has helped in determining “what kind of leader do I want to be,” she said, adding, “I want to help myself and others reach full potential. It’s about understanding each person’s motivations, what they want to try and learn, and making them part of the company ecosystem.”
Mammoth Biosciences is built on core technology Chen worked on at Doudna’s UC Berkeley lab. Chen earned her PhD as a graduate student researcher in this hotbed of innovation.
As a mentor, Doudna encouraged Chen to set up her own business upon graduation rather than to work at a major biotech company. “She told me I wasn’t shooting high enough,” said Chen, who has academic credentials from Harvard Medical School and Johns Hopkins Bloomberg School of Public Health, as well as an internship at a HIV research institute in Durban, South Africa.
“She’s a leader of the technical team and an overall strategist who has deep scientific knowledge and creativity, and can see where this technology is going,” said Doudna, whose UC Berkeley lab has been immersed in an ongoing patent battle over ownership of the biomedical technology. The U.S. Patent and Trademark Office recently determined in favor of the Broad Institute, a partnership of MIT and Harvard University. This decision impacts licensing for several CRISPR companies, but doesn’t extend to the particular gene editing system that Mammoth Biosciences uses. Doudna is also a co-founder of publicly traded CRISPR company Intellia Therapeutics.
At the age of 26, right after graduation, Chen had ventured out with fellow student and lab researcher Lucas Harrington to co-start a company. They set up shop at a biotech incubator in the up-and-coming Dogpatch neighborhood of San Francisco. “Janice and I split our time working in the lab and doing prototypes, and pitching venture capitalists,” recalled Harrington. Her husband, a scientist in San Francisco that she met at Johns Hopkins, “understands the journey” and devotion to starting this game-changing company. “It’s my life right now,” she said.
They met Mayfield partner Ursheet Parikh through a connection with Doudna. Parikh was advising Stanford PhD graduate Trevor Martin on launching a diagnostics testing start-up. The venture investor brought Martin, Doudna, Harrington and Chen together, and the team formed Mammoth Biosciences. Martin is CEO, Harrington is chief scientific officer, Doudna is chair of the Scientific Advisory Board while Chen is CTO.
“She’s a multi-faceted person and clearly a genius,” said Mayfield’s Parikh, a board member and serial investor in her company.
VC investing in gene editing reaches billions
Since 2014, CRISPR start-ups have attracted $3 billion in venture capital, according to Chris Dokomajilar, founder and CEO of biopharma database company DealForma. An analysis by GlobalData’s Pharma Intelligence Center shows 74 VC deals for CRISPR technology companies since 2012, with Mammoth Biosciences in the lead of most well-funded. The start-up has raised $265 million in four financings from at least 15 VC firms and angel investors.
The company’s work expanded rapidly during the pandemic in 2020. Among seven firms granted $249 million for rapid tests of Covid-19 from the National Institutes of Health, the firm scaled up its patented DetectR test for commercial labs diagnosing the virus. In a collaboration with GSK Consumer Healthcare in Warren, New Jersey, a handheld device that can perform rapid diagnostic tests of the coronavirus is being created. Additionally, Mammoth Biosciences teamed up in early 2021 with Agilent Technologies in Santa Clara to develop CRISPR testing systems for labs to expand and speed up detection of the coronavirus disease.
“She has a rare skill set to conceptualize the future and what this technology can do for humanity,” said another of her investors, Harsh Patel, co-founder and managing director at Wireframe Ventures. “She can turn incredible science in a lab into commercial technology products. It’s a big leap away from the lab.”
More developments came in rapid-fire sequence later in 2021 and into this year. Vertex Pharmaceuticals in Boston paid $41 million to the start-up to expand cell and genetic therapy tools, which could lead to $650 million in royalties. Bayer AG in Berlin paid $40 million to Mammoth Biosciences to focus on tests and cures for liver diseases, with royalties that could mount to $1 billion. Moreover, this January, the FDA granted the company emergency use authorization for a CRISPR-based molecular diagnostic testing of the coronavirus.
The accomplishments have tested Chen’s strength as an innovator and business leader, but investors say she is imperturbable. “I’ve never seen her frazzled in board meetings. She has strong opinions and she backs it up not by arguing, but by data,” said Omri Amirav-Drory, general partner at venture firm NFX, an investor and advisor. “I’m never selling my shares, I will give it to my kids. There’s a huge amount of IP in the company.”
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