Tag: crispr

A Gene-Edited Pig Kidney Was Just Transplanted Into a Person for the First Time

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Slayman received his first kidney transplant in 2018 from a human donor. The donor kidney initially functioned well, but Slayman started to go into kidney failure after years of living with diabetes. Diabetes is the leading cause of kidney disease, which can eventually result in kidney failure.

He had no choice but to go on dialysis, a treatment that removes excess fluid and waste from a person’s blood. But the dialysis caused complications—his blood vessels were clotting and failing. Slayman wound up in the hospital regularly and endured dozens of procedures to try to fix the problem.

“Slowly but surely, I witnessed my patient becoming increasingly despondent and depressed over his dialysis situation,” Winfred Williams, a kidney specialist and member of Slayman’s medical team, said on Thursday.

Finally, Williams suggested a pig kidney transplant. Slayman agreed. “I saw it not only as a way to help me, but a way to provide hope for the thousands of people who need a transplant to survive,” Slayman said in a statement released by Massachusetts General Hospital.

The procedure was performed under the Food and Drug Administration’s “compassionate use” pathway, which allows a patient with a life-threatening condition to access an experimental treatment when no other options exist. Slayman is also receiving an infusion of novel immunosuppressant drugs to prevent rejection of the organ. His medical team is currently monitoring his kidney function using ultrasound.

The Massachusetts team thinks the ideal candidate for a pig kidney will be a patient who was approved for a regular human kidney transplant but has a long wait time for a donor.

The pig kidney transplant comes on the heels of a procedure in January, in which surgeons at the University of Pennsylvania successfully attached a gene-edited pig liver to a braindead person and found that the organ functioned normally for 72 hours. The liver, also from eGenesis, contained the same 69 edits as Slayman’s kidney.

The liver is a more complicated organ because of the many functions it performs, so researchers don’t think pig livers are ready to be used in place of human ones just yet. Instead, they could be used outside the body and connected to patients who are waiting for a human organ or those who need temporary support while their own liver recovers.

Researchers have been working up to transplanting a modified pig kidney in a person. Last year, eGenesis reported that a kidney from one of its edited pigs functioned in a monkey for more than two years. And scientists at New York University and the University of Alabama at Birmingham have transplanted gene-edited pig kidneys into braindead patients to observe how well the organs function.

Jayme Locke, an abdominal transplant surgeon at the University of Alabama at Birmingham who has overseen some of those experiments, was thrilled to hear about the Boston kidney transplant. “This is wonderful news, and it’s great to see it move into the clinic,” she told WIRED in an interview.

Locke says the recent flurry of xenotransplantation experiments shows that the idea of using pig organs in people is gaining momentum and is here to stay. “I think it really has staying power and it’s going to really revolutionize the field and hopefully offer organs to all those in need,” she says.

Locke’s team is also looking to do pig-to-human kidney transplants. She said she has several patients in mind for the procedures and is just waiting on the FDA to give the greenlight. “We’re ready to go.”

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March 21, 2024
Bioengineering edible mycelium to enhance nutritional value, color, and flavor

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In a recent study published in Nature Communications, researchers developed a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives.

Study: Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit. Image Credit: Rattiya Thongdumhyu/Shutterstock.com

Background

Food production is estimated to account for a third of greenhouse gas emissions worldwide, contributing to biodiversity loss, environmental degradation, and new diseases.

Transitioning from industrial animal agriculture to alternatives is necessary to mitigate the planetary impact and sustainably feed the global population. Microbial food production offers improved safety and efficiency, more precise production control, and reduced animal suffering.

Filamentous fungi are a diverse group of microbes, including mushrooms and molds, and are highly advantageous for microbial food production.

Besides, their naturally high secretion capacity makes them potent hosts for protein production. In addition, owing to its filamentous structure that mimics the animal muscle structure, fungal biomass (mycelia) can be formulated into alternatives to meat (mycoprotein).

The study and findings

In the present study, researchers developed a modular synthetic biology toolkit for A. oryzae, a safe and edible fungus with a history of palatable consumption.

They created an alternative, easy-to-use clustered, regularly interspersed short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) approach, compatible with existing reagents.

This approach involved transforming CRISPR-Cas9 ribonucleoprotein complexes directly instead of encoding single-guide RNAs (sgRNAs) and Cas9 from a plasmid.

Moreover, the DNA template used to fix double-strand breaks contained an orotidine-5′-phosphate decarboxylase gene (pyrG) marker for positive and negative selection.

The system was designed such that a successful loop out of pyrG could only occur upon integrating the fixing template at the site of interest, wherein identical 300 bp sequences will flank it.

Ectopic integrations due to non-homologous end joining (NHEJ) in this system cannot loop out or survive on media with 5-fluoroorotic acid. A vital feature of this design was the recyclability of the pyrG marker upon insertion at the correct locus.

Further, candidate-neutral loci in A. oryzae were investigated to integrate genes for overexpression. The researchers explored the intergenic regions in the A. oryzae RIB40 genome and ranked the expression of two genes surrounding them.

A list of candidate loci predicted for high gene expression was generated, and ten regions were selected for further analysis.

Next, the team integrated green fluorescent protein (GFP) cassettes under the control of a strong, constitutive promoter (pTEF1) and examined fluorescence on the conidia of looped-out strains.

Of the ten loci, nine exhibited highly efficient integration, and GFP expression was detected from eight of these. All loci demonstrated higher expression than the positive control.

Next, the researchers aimed to establish a synthetic expression system (SES) in A. oryzae. To this end, they evaluated the ability of a characterized synthetic transcription factor (sTF) to drive the expression of mCherry from a core promoter (Cp).

They genetically integrated the sTF and induced a low basal expression under a Cp from A. niger. Separately, an mCherry cassette with 6x upstream activating sequences (UAS) was integrated at a different genomic location upstream of the Cp.

The team observed mCherry expression in conidia and mycelia. Both the sTF and UAS were required for the activity. Next, the team aimed to bioengineer an edible mycelium, focusing on the bioactive amino acid ergothioneine.

They speculated that its production could be increased by modulating the expression of endogenous ergothioneine biosynthetic genes in A. oryzae.

Orthologs of Egt1 and Egt2, enzymes from Neurospora crassa implicated in ergothioneine biosynthesis, were identified in A. oryzae.

The orthologs were then inserted at neutral loci; both genes were expressed under a bidirectional promoter or separately at different locations. Ergothioneine levels in the mycelium were low in RIB40, the background strain.

However, its levels were 11- and 21-fold elevated in bidirectional and separate promoter strains compared to RIB40. Ergothioneine levels in the bidirectional promoter stain were similar to those in oyster mushrooms. By contrast, its levels were 1.5-fold higher in the separate promoter strain.

There were no differences in protein content between engineered and wild-type strains. Nevertheless, a slight growth defect was observed with ergothioneine overproduction.

Next, the researchers applied these tools to enhance the sensory properties of the edible biomass. They targeted heme biosynthesis, as heme gives meat its (red) color and flavor upon cooking.

They identified potential heme biosynthetic genes in A. oryzae and targeted the expression of five predicted rate-limiting enzymes. Additionally, two copies of soy leghemoglobin were expressed as a potential heme sink, as high levels of free heme could be cytotoxic.

The biomass of the engineered strain was four-fold higher than that of the non-engineered strain.

Upon harvesting, the biomass was red compared to off-white in RIB40. This color difference persisted after cooking, enhancing the meat-like appearance of the fungal biomass.

The engineered mycoprotein contained all essential amino acids. Protein content or growth yield was not lower in the engineered strain.

Conclusions

The researchers developed a synthetic toolkit to integrate and regulate genes and pathways. They leveraged this toolkit and engineered A. oryzae mycoprotein to (over)produce ergothioneine at levels far greater than in natural dietary sources, i.e., mushrooms.

Additionally, the mycelia were engineered to overproduce heme for enhanced color and flavor. Notably, this work represents an early prototype; further evaluations of sensory attributes, food safety, consumer acceptance, and regulatory landscape are required.

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March 21, 2024
CRISPR could disable and cure HIV, suggests promising lab experiment

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An electron micrograph of HIV, which currently requires lifelong medication

Scott Camazine/Alamy Stock Photo

A new way to eradicate HIV from the body could one day be turned into a cure for infection by this virus, although it hasn’t yet been shown to work in people.

The strategy uses a relatively recent genetic technique called CRISPR, which can make cuts in DNA to introduce errors into viral genetic material within immune cells. “These findings represent a pivotal advancement towards designing a cure strategy,” researcher Elena Herrera Carrillo at the University of Amsterdam in the Netherlands said in a statement.

While infection with HIV was once nearly always fatal, those with the virus can now take drugs that stop it from reproducing. This gives them a nearly normal lifespan, as long as they diligently take their medicines every day.

But when people are first infected, some of the virus inserts its DNA into their immune cells, where it stays dormant. If they stop taking their HIV medicines, this DNA “reawakens” and the virus starts spreading through their immune systems again.

For a cure, we need some way of killing any dormant virus in the body. Several strategies have been tried, but none has so far been found to work.

The latest approach uses a gene-editing system called CRISPR. Originally discovered in bacteria, this homes in on a specific DNA sequence, making cuts in it. By changing the DNA sequence being targeted, the system can potentially be turned into a form of gene therapy for many conditions, with the first such treatment having been approved last year in the US and UK as a cure for sickle cell anaemia.

Several groups are investigating using CRISPR that targets a gene in HIV as a way of disabling dormant virus. Now, Carrillo and her team have shown that, when tested on immune cells in a dish, their CRISPR system could disable all virus, eliminating it from these cells. The work is due to be presented at the European Congress of Clinical Microbiology and Infectious Diseases in Barcelona, Spain, next month.

Jonathan Stoye at the Francis Crick Institute in London says that although the results are encouraging, the next step is trials in animals and eventually people to show the treatment can reach all the immune cells with dormant HIV. Some of these cells are thought to reside in bone marrow, but there may be other body sites involved too, he says. “There’s still a fair amount of uncertainty about whether there are other reservoirs in other parts of the body,” he says.

A Californian firm called Excision BioTherapeutics has previously shown that a CRISPR-based approach can reduce the amount of dormant virus in monkeys infected with a similar virus to HIV.

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March 19, 2024
Rapid, easy-to-use diagnostic test could save more lives from melioidosis

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Globally, more than half of patients die after infection with the neglected tropical disease, melioidosis, often before they are diagnosed. A new rapid test could save lives by diagnosing patients in hours, rather than several days taken by current bacterial culture methods, meaning they receive the correct antibiotics faster.

The test uses CRISPR to detect a genetic target that is specific to Burkholderia pseudomallei, the bacterium that causes melioidosis, with 93 per cent sensitivity. It was developed by researchers at the Mahidol-Oxford Tropical Medicine Research Unit (MORU), Chiang Mai University, Vidyasirimedhi Institute of Science and Technology (VISTEC) in Thailand, and the Wellcome Sanger Institute in the UK.

The results, published today (14 March) in Lancet Microbe, mean more lives could be saved from melioidosis, with a rapid, easy-to-use diagnostic test that could be rolled out globally.

Melioidosis is a neglected tropical disease that is estimated to affect 165,000 people worldwide each year, of whom 89,000 die from the disease. It is caused by the bacterium, Burkholderia pseudomallei, which lives in soil and water in tropical and subtropical regions, and enters human bodies via inoculation through skin abrasions, ingestion or inhalation.

It is difficult to diagnose melioidosis as symptoms vary from localised abscess or pneumonia to acute septicaemia, or may present as a chronic infection. As a result of this, and the locations of isolated communities in rural areas that it mostly affects, the disease remains hugely underreported.

Currently, melioidosis is diagnosed in patients after bacterial samples are cultured, which takes three to four days. In Thailand, approximately 40 per cent of patients with melioidosis die, many of whom die within the first one to two days following admission to hospital, while waiting for a diagnosis.

There is no licensed vaccine for melioidosis, but patients can be effectively treated with intravenous antibiotics – ceftazidime or carbapenem – during the first intensive phase of treatment. However, current practices often involve initially treating patients with a range of unnecessary antibiotics to target the various symptoms the disease produces, which can waste time and resources.

In a new study, the team set out to develop a new rapid test to reduce the time taken to correctly diagnose and treat patients with melioidosis.

The researchers identified a genetic target specific to B. pseudomallei by analysing over 3,000 B. pseudomallei genomes, most of which were sequenced at the Sanger Institute. They searched for conserved regions of the genome and screened the targets against other pathogens and human host genomes, to ensure their chosen target was specific to B. pseudomallei.

Their test, called CRISPR-BP34, involves rupturing bacterial cells and using a recombinase polymerase amplification reaction to amplify the bacterial target DNA for increased sensitivity. Additionally, a CRISPR reaction is used to provide specificity, and a simple lateral flow ‘dipstick’ read-out is employed to confirm cases of melioidosis.

To assess the efficacy of the test, the team collected clinical samples from 114 patients with melioidosis and 216 patients without the disease at Sunpasitthiprasong Hospital, a hospital in northeast Thailand where melioidosis is endemic. The CRISPR-BP34 test was then applied to these samples.

The new test showed enhanced sensitivity at 93 per cent, compared to 66.7 per cent in bacterial culture methods. It also delivered results in less than four hours for urine, pus, and sputum samples, and within one day for blood samples. This is a significant improvement over the current bacterial culture diagnostic method, which typically takes three to four days.

This new rapid diagnostic test will enable health professionals to prescribe the correct antibiotics faster, meaning fewer patients will die while waiting for a diagnosis. While saving precious time, the new test will also save resources and money, with fewer unnecessary antibiotics prescribed and less time for patients in hospital.

In next steps for the team, they are currently designing randomized clinical trials to show the effectiveness of these tests in hospital settings. Plus, members of the team will begin investigating the role of human genetics in susceptibility and immune response to melioidosis infection.

Dr Claire Chewapreecha, co-lead author at the Mahidol-Oxford Tropical Medicine Research Unit (MORU), Thailand, and Wellcome Sanger Institute International Fellow, said: “Working in rural Thailand has many limitations. But we have shown that limitations breed innovation, and what succeeds here can succeed anywhere. I am so proud of the team behind this new, robust rapid diagnostic test for melioidosis, and hope that it can potentially be used anywhere in the world to get the right treatments to patients faster, ultimately saving lives.”

Dr Somsakul Wongpalee, co-lead author at Chiang Mai University, Thailand, said: “We carefully designed the rapid diagnostic test based on CRISPR-BP34, with a robust algorithm, and tested its performance in vitro. We are thrilled that the CRISPR-BP34 test demonstrates outstanding diagnostic efficacy when tested on clinical samples, showcasing its potential to significantly impact patient outcomes and potentially save lives in the near future.”

This research is a testament to international collaboration and how the application of genomics at scale leads to clinical intervention. Using a genetic target mined from a bank of thousands of bacterial genomes, the team was able to produce an incredibly sensitive test that is specific to the bacterium behind melioidosis. I look forward to seeing the clinical impacts of this research.”

Professor Nick Thomson, Senior Author and Head of Parasites and Microbes at the Wellcome Sanger Institute

Professor Nick Day, senior author and Director of the Mahidol-Oxford Tropical Medicine Research Unit (MORU), Thailand, and the Wellcome Trust Thailand Asia and Africa Programme, said: “Melioidosis has been neglected despite its high mortality rate and high incidence in many parts of Asia. Early diagnosis is essential so that the specific treatment required can be started as soon as possible. The new rapid diagnostic tool developed through this collaboration has the potential to be a game-changer.”

Source:

Wellcome Trust Sanger Institute

Journal reference:

Pakdeerat, S., et al. (2024) Benchmarking CRISPR-BP34 for point-of-care melioidosis detection in low-income and middle-income countries: a molecular diagnostics study. The Lancet Microbe. doi.org/10.1016/S2666-5247(23)00378-6.

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March 14, 2024
Plant-killing genetic technology could wipe out superweeds

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A ‘gene drive’ that spreads through plant populations could be used to wipe out pests such as superweeds, or to help save species by making them resistant to heat or disease

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March 13, 2024
Scientists develop robust iPSC-derived rejuvenated T lymphocytes for cervical cancer treatment

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Cervical cancer is among the most common malignancies affecting women worldwide. In 2020 alone, approximately 600,000 women were diagnosed with this disease, and over 314,000 died from it. In 99% of the cases, cervical cancer cells harbor human papilloma virus (HPV), and thus, HPV vaccines are an effective way to mitigate the risk of developing this disease. Unfortunately, such preventive measures are useless against established cancers, which are generally incurable once they become metastatic or relapsing.

Fortunately, scientists have made substantial progress in developing a promising therapeutic strategy for cervical cancer: rejuvenated cytotoxic T lymphocytes (rejTs). These lymphocytes can be engineered to target HPV-specific antigens that are expressed predominantly in cervical cancer cells, constituting a type of targeted immunotherapy. Ideally, rejTs would be produced from induced pluripotent stem cells (iPSCs) gathered from the patient themselves. However, this process is not clinically feasible in terms of both time and cost.

Against this backdrop, a research team including Chief Professor Miki Ando, graduate student Yoshiki Furukawa, and Assistant Professor Midori Ishii from Juntendo University School of Medicine, Japan, has recently achieved a breakthrough by developing robust iPSC-derived rejTs for cervical cancer treatment. Their work was published online on December 12, 2023, in Cell Reports Medicine.

The team sought to address one of the key roadblocks for allogeneic iPSC-derived rejTs, which refers to rejTs produced from ‘standardized’ iPSCs rather than derived from the patient’s cells. Prof. Ando explains, “In immunocompetent cervical cancer patients, the dominant problem is the rejection of foreign T lymphocytes by the recipient’s CD8+ T lymphocytes or natural killer (NK) cells.” The patient’s immune system tends to attack the therapeutic HPV-specific rejTs before they can neutralize cancer cells.

To overcome this issue, the research team used CRISPR-Cas9 two-step “scarless” gene editing on iPSCs derived from an HPV-specific cytotoxic T lymphocyte clone. The first modification was the deletion of all HLA class I antigens from the cells. The role of these surface protein is to present peptides to CD8+ T lymphocytes, which promptly eliminate any cells displaying foreign or anomalous peptides. After the modification, the cells are essentially able to evade detection by CD8+ T lymphocytes.

The second modification involved introducing the limited expression of two specific HLA antigens, namely HLA-A24 and HLA-E. This enabled the engineered cells to evade attacks from NK cells, which specifically target cells lacking these surface antigens. By selecting HLA-A24, the engineered cells are naturally compatible with a significant portion of South American, Eastern Asian, North American, and European populations.

After implementing these modifications using CRISPR-Cas9, the researchers induced the differentiation of the iPSCs into T lymphocytes and tested their ability to fight cervical cancer cells while evading allogeneic immune responses both in vitro and in vivo. These experiments yielded very promising results, as the generated rejTs did not trigger attacks from either CD8+ T cells or NK cells while simultaneously achieving strong cytotoxicity against tumor cells.

Compared to the control group, mice engrafted with cervical cancer cells and injected with the gene-edited rejTs survived longer and exhibited significantly reduced tumor sizes and proliferation index. To gain further insights into the enhanced therapeutic effects of the rejTs, the researchers conducted single-cell RNA sequencing analyses. These analyses revealed that the population of rejTs was highly enriched with tissue resident memory T cells, which establish residence in the mucosa of the cervix and provide stronger protection. Prof. Ando remarks, “We found that the enhanced cytotoxicity against cervical cancer occurred through TGFβ signaling and increased CD103 expression.“

Taken together, the results of this study showcase a powerful method to make iPSC-derived rejTs a viable option to treat one of the most common forms of cancer. Prof Ando concludes, “The HLA-engineered HPV-rejTs obtained using our method provide a sustainable and promising approach toward successful ‘off-the-shelf’ T cell therapy, which could help in overcoming cervical cancer. We are planning on conducting an investigator-initiated clinical trial in 2024.“

Source:

Juntendo University Research Promotion Center

Journal reference:

Furukawa, Y., et al. (2023). iPSC-derived hypoimmunogenic tissue resident memory T cells mediate robust anti-tumor activity against cervical cancer. Cell Reports Medicine. doi.org/10.1016/j.xcrm.2023.101327.

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March 6, 2024
If gene-edited meat eases the climate crisis, then we must embrace it

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EATING meat is a major contributor to two of the greatest problems humanity faces: global warming and the loss of biodiversity. Farming is one of the main sources of greenhouse gas emissions, while the amount of land turned over to grow food for livestock leaves less space for wildlife.

This is why many studies have highlighted the massive environmental benefits there would be if only people ate less meat. A plant-based diet has a much lower footprint in terms of carbon emissions and land. What’s more, there are also ethical and health arguments for such a diet.

Yet global meat…

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February 28, 2024
Eye movement reflex reveals genetic association with autism

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Scientists at UC San Francisco may have discovered a new way to test for autism by measuring how children’s eyes move when they turn their heads.

They found that kids who carry a variant of a gene that is associated with severe autism are hypersensitive to this motion.

The gene, SCN2A, makes an ion channel that is found throughout the brain, including the region that coordinates movement, called the cerebellum. Ion channels allow electrical charges in and out of cells and are fundamental to how they function. Several variants of this gene are also associated with severe epilepsy and intellectual disability.

The researchers found that children with these variants have an unusual form of the reflex that stabilizes the gaze while the head is moving, called the vestibulo-ocular reflex (VOR). In children with autism, it seems to go overboard, and this can be measured with a simple eye-tracking device.

The discovery could help to advance research on autism, which affects 1 out of every 36 children in the United States. And it could help to diagnose kids earlier and faster with a method that only requires them to don a helmet and sit in a chair.

“We can measure it in kids with autism who are non-verbal or can’t or don’t want to follow instructions,” said Kevin Bender, PhD, a professor in the UCSF Weill Institute for Neurosciences and co-senior author of the study, which appears Feb. 26 in Neuron. “This could be a game-changer in both the clinic and the lab.”

A telltale sign of autism in an eye reflex

Of the hundreds of gene mutations associated with autism, variants of the SCN2A gene are among the most common.

Since autism affects social communication, ion channel experts like Bender had focused on the frontal lobe of the brain, which governs language and social skills in people. But mice with an autism-associated variant of the SCN2A gene did not display marked behavioral differences associated with this brain region.

Chenyu Wang, a UCSF graduate student in Bender’s lab and first author of the study, decided to look at what the SCN2A variant was doing in the mouse cerebellum. Guy Bouvier, PhD, a cerebellum expert at UCSF and co-senior author of the paper, already had the equipment needed to test behaviors influenced by the cerebellum, like the VOR.

The VOR is easy to provoke. Shake your head and your eyes will stay roughly centered. In mice with the SCN2A variant, however, the researchers discovered that this reflex was unusually sensitive. When these mice were rotated in one direction, their eyes compensated perfectly, rotating in the opposite direction. 

But this increased sensitivity came at a cost. Normally, neural circuits in the cerebellum can refine the reflex when needed, for example to enable the eyes to focus on a moving object while the head is also moving. In SCN2A mice, however, these circuits got stuck, making the reflex rigid.

A mouse result translates nearly perfectly to kids with autism

Wang and Bender had uncovered something rare: a behavior that arose from a variant to the SCN2A gene that was easy to measure in mice. But would it work in people?

They decided to test it with an eye-tracking camera mounted on a helmet. It was a “shot in the dark,” Wang said, given that the two scientists had never conducted a study in humans.

Bender asked several families from the FamilieSCN2A Foundation, the major family advocacy group for children with SCN2A variants in the US, to participate. Five children with SCN2A autism and eleven of their neurotypical siblings volunteered.

Wang and Bender took turns rotating the children to the left and right in an office chair to the beat of a metronome. The VOR was hypersensitive in the children with autism, but not in their neurotypical siblings.

The scientists could tell which children had autism just by measuring how much their eyes moved in response to their head rotation.

A CRISPR cure in mice

The scientists also wanted to see if they could restore the normal eye reflex in the mice with a CRISPR-based technology that restored SCN2A gene expression in the cerebellum.

When they treated 30-day-old SCN2A mice – equivalent to late adolescence in humans – their VOR became less rigid but was still unusually sensitive to body motion. But when they treated 3-day-old SCN2A mice – early childhood in humans – their eye reflexes were completely normal.

These first results, using this reflex as our proxy for autism, point to an early window for future therapies that get the developing brain back on track.”

Chenyu Wang, UCSF graduate student

It’s too early to say whether such an approach might someday be used to directly treat autism. But the eye reflex test, on its own, could clear the way to more expedient autism diagnosis for kids today, saving families from long diagnostic odysseys.

“If this sort of assessment works in our hands, with kids with profound, nonverbal autism, there really is hope it could be more widely adopted,” Bender said.

Source:

University of California – San Francisco

Journal reference:

Wang, C., et al. (2024). Impaired cerebellar plasticity hypersensitizes sensory reflexes in SCN2A-associated ASD. Neuron. doi.org/10.1016/j.neuron.2024.01.029.

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February 28, 2024
First gene-edited meat will come from disease-proof CRISPR pigs

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Gene-edited pigs are protected from porcine reproductive and respiratory syndrome

Genus PIC

Pigs that are immune to a disease estimated to cost farmers $2.7 billion a year globally look set to become the first genetically modified farm animals to be used for large-scale meat production.

“We could very well be the first,” says Clint Nesbitt of international breeding company Genus, which has created hundreds of the CRISPR-edited pigs in preparation for a commercial launch.

He is confident that the animals,…

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February 23, 2024
Identification of genetic determinants of micronucleus formation

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In a recent study published in Nature, researchers identified the genetic determinants of micronuclei formation (MN).

Study: Genetic determinants of micronucleus formation in vivo. Image Credit: Dimarion/Shutterstock.com

Background

Genomic instability and extracellular MN accumulation are hallmarks of various disorders, including inflammation-related diseases, cancer, and aging. MN are fragments of chromosomes formed due to errors in mitotic segregation or unrepaired DNA breaks.

MN are protected by an atypical nuclear envelope and can persist for several cellular generations, acquire aberrant epigenetic marks, and replicate their DNA.

Further, the nuclear envelope of MN can rupture, resulting in the accumulation of MN DNA damage, chromosomal recombination, and a potent pro-inflammatory response that could lead to cellular senescence.

The study and findings

The present study investigated the factors regulating MN formation in vivo. They screened over 6,000 mice across 997 loss-of-function mutant lines using a detection method that enumerated MN in erythrocytes.

This defined genes upon which disruption decreased (-MN) or increased (+MN) the formation and accumulation of MN relative to wild-type (WT) controls. Hits were grouped into three tiers (1 – 3) based on statistical significance.

Tier 1 contained 27 -MN and 29 +MN genes at p < 0.001, tier 2 included 26 -MN and 23 +MN genes at p < 0.005, and tier 3 comprised 21-MN and 19 +MN genes at p < 0.01.

Next, the team selected seven -MN genes (ABCB6, DUSP7, JMJD1C, HMX3, KLK9, PIAS2, and TATDN3) from tier 1 and disrupted them using clustered regularly interspersed short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) editing in human CHP-212 cells.

Cells were subjected to a low, chronic hydroxyurea dose that increased the average basal rate of MN from 1.5% to 5% in WT cells.

MN frequency in DNA topoisomerase III alpha (TOP3A)-knockout (KO) and DNA replication and sister chromatid cohesion 1 (DSCC1)-KO positive controls was 26.04% and 9.6%, respectively.

Decreased micronucleation was expectedly observed after disrupting protein inhibitor of activated stat 2 (PIAS2) (2.91%), dual specificity phosphatase 7 (DUSP7) (2.19%), and TatD DNase domain containing 3 (TATDN3) (1.31%).

Further, phenotyping analyses of all mouse mutant lines revealed a role for screened hits in maintaining homeostasis, with several lines exhibiting phenotypes associated with higher mortality and metabolic, neuronal, skeletal, and immune dysfunction.

In addition, the researchers integrated these findings with the loss-of-Y (LOY) genome-wide association study (GWAS) to assess the human relevance of MN screen hits.

LOY loci were enriched for human orthologs of MN genes. Besides, MN-linked genes were established as disease-associated or contributors to tumorigenesis.

Among the +MN group, a class of genes comprised factors involved in sister chromatid cohesion and defects in human orthologs of these genes result in multiorgan syndromes known as cohesinopathies.

The team investigated the role of DSCC1 (one of the genes from this class) in human disease by analyzing data from the United Kingdom (UK) Biobank.

They observed common genetic variants associated with bone mineral density (BMD) and body mass index that seemingly conferred their effects through altered DSCC1 expression.

Further, rare protein-truncating DSCC1 variants also showed suggestive associations with BMD (independent of common variants). The researchers characterized the Dscc1-mutant mice because this mutant had the most significant MN increase.

Dscc1-mutant (Dscc1^-/-) mice were sub-viable at embryonic day 14.5 (E14.5). Surviving mutants had higher body weight, skeletal abnormalities, higher bone mineral content, and testicular atrophy.

Cultures of Dscc1^-/-mouse embryonic fibroblasts (MEFs) isolated from E13.5 embryos showed slower growth and higher genomic instability than those from WT littermates (Dscc1^+/+).

Extensive chromosome breakage and re-arrangement events were also observed in Dscc1^-/- MEFs. Dscc1-mutants exhibited significantly reduced tumor latency, suggesting that DSCC1 can act as a tumor suppressor.

DSCC1 disruption through knockdown (KD) in human induced pluripotent stem (iPS) cells caused destabilization of the two other components of the alternative replication factor C complex (RFC^CTF18). They increased the abundance of DNA damage response proteins.

Further, a genome-wide CRISPR-Cas9 screen was performed for genes that affect the proliferation of DSCC1-deficient human iPS cells.

This revealed four genes (phenotype suppressors), which could (when disrupted) partially rescue the proliferation defect, and five genes (phenotype enhancers), which (when disrupted) further reduced their fitness/proliferation.

The team focused on the relationship between DSCC1 and sirtuin 1 (SIRT1), one of the phenotype suppressors. They observed that SIRT1 KO in HEK293 cells partially rescued the cell viability defects caused by DSCC1 depletion.

Treating DSCC1-mutant iPS cells with selisistat, a selective SIRT1 inhibitor, partially rescued cellular proliferation and reduced MN formation.

Conclusions

Taken together, the researchers identified over 100 genes related to MN formation. These included Dscc1^-/-, a semi-viable mutant with neurologic, developmental, structural, reproductive, and skeletal anomalies and tumor predisposition.

Disruption of DSCC1 caused a loss of cellular viability that was partially rescued by inhibiting SIRT1.

These data are collectively a resource of genetic determinants of genomic instability and provide a platform to identify functional/genetic modifiers relevant to human disease.

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February 19, 2024