Tag Archives: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

Gene-editing tools like CRISPR unlock promising new topical treatments for the first time for skin diseases

Exciting? Yes. However, as far as I can tell, hey are a long way from human clinical trials. Onto the good news, from a January 27, 2026 University of British Columbia (UBC) news release (can be found here at the Life Science Institute, here at the Faculty of Medicine, and here at the university’s press office), Note: Links have been removed,

Gene-editing tools like CRISPR (clustered regularly interspaced short palindromic repeats) have unlocked new treatments for previously uncurable diseases. Now, researchers at the University of British Columbia are extending those possibilities to the skin for the first time.

The UBC team, together with researchers from the Berlin Institute of Health at Charité in Germany, has developed the first gene therapy capable of correcting faulty genes when applied directly to human skin. Outlined today [January 27, 2926] in a paper published in Cell Stem Cell, the breakthrough could lead to new treatments for a wide range of genetic skin conditions, from rare inherited diseases to more common disorders like eczema.

“With this work, we show that it is possible to correct disease-causing mutations in human skin using a topical treatment that is safe, scalable and easy-to-use,” said Dr. Sarah Hedtrich, an associate professor at UBC’s school of biomedical engineering and senior author of the study. “Importantly, the approach corrects the root cause of disease, and our data suggests that a one-time treatment might even be enough to provide a lasting and durable cure.”

Broad therapeutic potential 

In the study, the researchers show the gene therapy can correct the most common genetic mutation behind autosomal recessive congenital ichthyosis (ARCI), a rare and life-threatening inherited skin disorder that appears at birth.

Affecting approximately one in 100,000 people, ARCI causes lifelong complications including extremely dry and scaly skin, chronic inflammation and a high risk of infections. There is currently no cure or effective treatment, and patients must manage their symptoms for life.

“For many patients, this condition is not only physically painful, but also deeply stigmatizing and isolating,” said Dr. Hedtrich.

By testing the treatment in models made from living human skin, the team showed it can restore up to 30 per cent of normal skin function—a level that previous research suggests could be clinically meaningful for returning skin function to normal.

While ARCI affects relatively few people, the researchers say the treatment strategy could be adapted to many other genetic skin diseases, including epidermolysis bullosa—a severe skin blistering condition often called ‘butterfly skin’—and potentially more common conditions such as eczema or psoriasis.

“The approach we developed is a platform technology,” said Dr. Hedtrich. “It can be readily adapted to treat almost any skin disease.”

A new way to deliver CRISPR gene editing 

Despite major advances in gene editing, applying the technology to skin diseases has remained a long-standing challenge. The skin’s primary role is to protect the body from the outside world, making it difficult to deliver large biological therapies, such as gene editors, past its protective barrier.

To overcome this, the team developed a novel delivery method that uses lipid nanoparticle technology, or LNPs. These microscopic “bubbles of fat,” pioneered by UBC professor Dr. Pieter Cullis [emphasis mine] and brought to global prominence through mRNA vaccines, are able to carry gene-editing technology into cells.

Using a clinically approved laser, the researchers first create microscopic, pain-free openings in the outer layers of the skin. This allows the lipid nanoparticles to pass through the skin barrier and reach skin stem cells beneath the surface. Once inside, the gene editor corrects the underlying DNA mutation, enabling the skin to begin functioning more normally.

“This is a highly targeted, localized approach,” said Dr. Hedtrich. “The treatment stays in the skin and we saw no evidence of off-target effects, which is a critical safety milestone.”

The study was conducted in close collaboration with Vancouver-based biotech company NanoVation Therapeutics [emphasis mine], a UBC spin-off focused on developing LNP-based genetic medicines. The researchers now hope to bring the treatment into clinical testing and have already been working with regulatory authorities to define the necessary safety and efficacy studies. [emphasis mine]

“Our goal now is to take this from the lab into first-in-human clinical trials,” said Dr. Hedtrich. “We hope this work will ultimately lead to a safe, effective treatment that can transform the lives of patients who currently have no real therapeutic options.”

Here’s a link to and a citation for the paper,

Lipid nanoparticle-based non-viral in situ gene editing of congenital ichthyosis-causing mutations in human skin models by Dilem Ceren Apaydin, Gaurav Sadhnani, Tiffany Carlaw, Jan Renziehausen, Elena Lizunova, Viviane Filor, Anna Hiller, Sophia Brumhard, Vincent Halim, Ulrike Brüning, Johannes Bischof, Rafaela Horbach Marodin, Daniel Z. Kurek, Manuel Rhiel, Sandra Ammann, Tatjana I. Cornu, Toni Cathomen, Leif Erik Sander, Benedikt Obermayer, Fabian Coscia, Jennifer Kirwan, Ulrich Koller, Achim D. Gruber, Wolfgang Bäumer, and Sarah Hedtrich. Cell Stem Cell Available online 27 January 2026 In Press, Corrected Proof DOI: https://doi.org/10.1016/j.stem.2026.01.001

This paper is behind a paywall.

NanoVation Therapeutics, the UBC spin-off mentioned in the news release, was co-founded by Pieter Cullis according to the company’s Our Team webpage.

Ready to start eating gene-edited pork?

Ready or not, it’s coming to our grocery stores. Last year’s September 19, 2025 posting “Gene-edited pork coming to Canadian consumers—soon (maybe)” now has its companion piece. A January 23, 2026 Health Canada news release announces approval,

Canada approves pigs resistant to porcine reproductive and respiratory syndrome viruses for use in food and feed

Porcine reproductive and respiratory syndrome viruses (PRRSV) are one of the most devastating classes of viruses affecting farmed pigs, representing significant losses for Canada’s producers and higher prices at the store for Canadian consumers.

Today, Health Canada and the Canadian Food Inspection Agency (CFIA) announced the completion of independent, comprehensive safety assessments on pigs resistant to PRRSV following applications from Genus PLC [sic] and PIC [Pig Improvement Company] Canada, Ltd., respectively. Health Canada has concluded that foods made from these pigs are as safe and nutritious for people to eat as pork currently available in Canada. The CFIA has confirmed that these pigs are also safe and effective for use in livestock feeds.

Improving resistance to PRRSV in pigs will help farmers avoid illness in their herds from these viruses, reduce antibiotic use, and improve animal welfare, while supporting a more stable, affordable and sustainable food supply.

Genus PLC’s PRRSV-resistant pigs are already permitted for food use in the U.S., Brazil, Colombia and the Dominican Republic. Genus PLC has indicated that even though Canada has approved the sale of PRRSV resistant pigs, the company does not intend to sell these pigs before further regulatory authorization in other key markets. The company and Health Canada are committed to the highest level of transparency and will communicate to the public when this new technology enters the Canadian market.

While genetically engineered foods are considered as safe and nutritious as conventional foods, the Government of Canada recognizes that information about genetic engineering is important. That is why we have been working with the Canadian General Standards Board since November [2025?] on a public review of the National Standard for labelling and advertising of foods that are, and are not, products of genetic engineering. The CFIA uses this Standard to provide its guidance to companies on the labelling of foods.

Quick facts

  • In 2024, the swine industry generated over $6.3 billion in farm cash receipts and Canada produced 2.34 million tonnes of pork and exported 1.45 million tonnes of pork.
  • As of January 1, 2025, there were 13.9M hogs on 6,885 Canadian farms.
  • PRRSV infections can cause severe symptoms in pigs like breathing problems, fever, the inability to eat, and can lead to stillborn piglets and death.
  • There are currently no effective treatments, and vaccination has only been partially effective.
  • In Canada, food labelling is required when there are well-established health risks or significant changes to the nutritional qualities of the food. For example, an allergen in a food must be labelled to alert consumers of the risk.
  • Because Health Canada found no health and safety concerns, no special labelling is required for foods from these PRRSV-resistant pigs.
  • In addition to the assessments noted above, in December 2025, the New Substances program—jointly administered by Environment and Climate Change Canada and Health Canada—determined that environmental and human health risks from indirect exposure to these pigs is no different from pigs currently available in Canada.

Amina Zafar’s January 23, 2026 article of Canadian Broadcasting News (CBC) news online offers, in addition to information from the news release, detail about a previous attempt to introduce genetically modified food to Canadians, Note: Links have been removed,

Food made from pigs engineered to be resistant to a porcine virus are as safe and nutritious to eat as pork currently on the market, Health Canada said Friday.

Federal regulators announced pigs resistant to porcine reproductive and respiratory syndrome viruses (PRRSV) — which it called a devastating class of viruses in farmed pigs — are safe to use in food and livestock feeds.

More than 25 years ago, scientists at Memorial University in Newfoundland developed a salmon that would become the first transgenic food animal approved for sale in Canada. Sold as AquAdvantage Salmon, the DNA came from Pacific salmon and the eel-like ocean pout.

At the time, the Council of Canadians said it was concerned about the amount of water extracted, effluent and transport of eggs between facilities.

Unlike the genetically modified salmon, the PRRSV-resistant pigs were created using CRISPR gene-editing technology that makes precise genetic changes without introducing genetic material from another species, scientists say. 

“This is the first gene-edited animal that has been approved for commercial sale in Canada,” said Gwendolyn Blue, a professor in the geography department at the University of Calgary who studies the politics and values surrounding gene editing.

While Blue recognizes the advantages PRRSV-resistant pigs could bring, she’s also notes there are ethical, moral, cultural and political questions surrounding the approval, such as how assessments about safety were made, as well as Canada’s updated biotechnology regulations.

“One of the concerns that people have had within a Canadian context is labeling,” she said. “There is no mandatory labeling and this is something that is currently under review.”

While there is a Winnipeg-based PIC (Pig Improvement Company) branch, the North American offshoot of the company (UK’s Genus plc) is headquartered in the US state of Tennessee. I quite appreciate the detail offered in the PIC January 23, 2026 news release,

Today Health Canada and the Canadian Food Inspection Agency (CFIA) approved PIC’s PRRS-resistant pigs for sale as food in Canada after rigorous and thorough review. Additionally, Environment & Climate Change Canada (ECCC) conducted its own review of the research and data and concluded that PRRS-resistant pigs may now be manufactured [?] or imported in Canada.

“We have spent years conducting extensive research, validating our findings and working with the Canadian government to gain approval,” said Matt Culbertson, PIC’s Chief Operating Officer. “Today marks a major milestone for consumers, farmers, and the entire pork industry who have hoped for relief from PRRS for decades.”

Review of Research and Data Confirms Safety

Health Canada and CFIA conducted scientific assessments that ensured the pigs are safe for consumption and do not differ from other pigs available on the market. Their evaluation led them to conclude that aside from the precise deletion of a small part of a gene involved in PRRS infection, the PRRS-resistant pigs are no different than other pigs.

Health Canada and CFIA further concluded that there are no differences in the nutritional value of the pork from PRRS-resistant pigs compared to other pork available for consumption.

How Removing Animal Disease Can Improve Animal Welfare

Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most devastating, global swine diseases. It causes needless suffering and premature death for pigs, negatively impacts animal welfare, exacerbates the need for antibiotics and increases the environmental impact of raising pigs.

“Addressing PRRS can allow us to improve animal welfare, reduce the need for antibiotics and decrease the environmental impact of raising pigs,” said Todd Wilken, PIC Director of North America Sales. “In fact, recent research indicates that PRRS increases the need for antibiotics by more than 2 times compared to pigs without PRRS.1

PIC Has Earned Regulatory Clearance in a Number of Countries

Canada joins a growing list of countries that have deemed the gene edit safe and effective. The U.S. FDA approved the gene edit in April 2025, while Colombia, Brazil, Dominican Republic and Argentina have issued positive determinations for PRRS-resistant pigs, meaning those countries have recognized that the pigs are not GMO and should be treated the same as any other pigs.

“We are committed to the responsible and intentional introduction of the PRRS-resistant pig around the globe. Gaining approval in Canada is an important step in this process, and we are working with additional countries to gain regulatory approval and protect global trade prior to initiating sales and delivery,” said Culbertson.

PIC is a wholly owned subsidiary of Genus, a UK-based company, publicly traded on the London Stock Exchange.

Canadian consumers indicate above average purchase likelihood for pork from gene-edited pigs

Research conducted by Circana [US market research company] and commissioned by PIC in late 2025 found that there is an above average likelihood that Canadian consumers will purchase pork from gene-edited pigs.

The research polled more than 5,000 pork consumers from eight key countries, representative across genders and ages 18 to 70.

Circana found that 90% of Canadian consumers are open to purchasing pork from gene-edited pigs and that responsibly reducing the need for antibiotics ranked as the top motivator.

Visit PRRSResistantPig.com for more information.

About PIC  

PIC (Pig Improvement Company) is the global leader in swine genetics. PIC provides genetically superior breeding stock to pig producers and supports them with technical services to help them realize genetic potential. PIC is a subsidiary of Genus, a UK-based company with a vision to pioneer animal genetic improvement to help nourish the world. www.pic.com

About Genus

Genus is a world-leading animal genetics company, supplying high-quality breeding animals with desirable characteristics to farmers, and enabling them to produce better quality meat and milk more efficiently to feed the world more sustainably. PIC is the porcine division within Genus. www.genusplc.com

Sources:

  1. Isadora Machado, Thomas Petznick, Ana Paula S. Poeta Silva, Chong Wang, Locke Karriker, Daniel C.L. Linhares, Gustavo S. Silva. “Assessment of changes in antibiotic use in grow-finish pigs after the introduction of PRRSV in a naïve farrow-to-finish system.” Preventive Veterinary Medicine, 2024. https://doi.org/10.1016/j.prevetmed.2024.106350

A bit disturbing, the change in terminology, that is. Since when do farmers manufacture pigs? Don’t they breed and/or grow pigs?

As for Canadians, this gene-edited pork will not be labelled under the current rules. Although that could change it may take a while as the government of Canada’s “Canadian General Standards Board conducts, since November [2025?], a public review of the National Standard for labelling and advertising of foods that are, and are not, products of genetic engineering.”

Gene editing to identify and change parts of chicken DNA and limit the spread of bird flu virus

This news comes from the University of Edinburgh (Scotland). From an October 10, 2023 news item on phys.org, Note: A link has been removed,

Scientists have used gene editing techniques to identify and change parts of chicken DNA that could limit the spread of the bird flu virus in the animals.

Researchers were able to restrict—but not completely block—the virus from infecting chickens by altering a small section of their DNA.

The birds showed no signs that the change in their DNA had any impact on their health or well-being.

The findings are an encouraging step forward, but experts highlight that further gene edits would be needed to produce a chicken population which cannot be infected by bird flu—one of the world’s most costly animal diseases.

An October 10, 2023 University of Edinburgh press release, which originated the news item, provides more detail about this research,

Gene editing

Scientists from University of Edinburgh, Imperial College London and the Pirbright Institute bred the chickens using gene editing techniques to alter the section of DNA responsible for producing the protein ANP32A. During an infection, flu viruses hijack this molecule to help replicate themselves.

When the ANP32A gene-edited chickens were exposed to a normal dose of the H9N2-UDL strain of avian influenza virus – commonly known as bird flu – 9 out of 10 birds remained uninfected and there was no spread to other chickens.

Partial protection

The research team then exposed the gene-edited birds to an artificially high dose of avian influenza virus to further test their resilience.

When exposed to the high dose, half of the group – 5 out of 10 birds – became infected. However, the gene edit did provide some protection, with the amount of virus in the infected gene-edited chickens much lower than the level typically seen during infection in non-gene-edited chickens.

The gene edit also helped to limit onward spread of the virus to just one of four non-gene-edited chickens placed in the same incubator. There was no transmission to gene-edited birds.

Viral evolution

Scientists found that in the ANP32A gene-edited birds, the virus had adapted to enlist the support of two related proteins – ANP32B and ANP32E – to replicate.

Following lab tests, scientists found that some of the mutations enabled the virus to utilise the human version of ANP32, but its replication remained low in cell cultures from the human airway.

Experts say that additional genetic changes would be needed for the virus to infect and spread effectively in humans.

However, the findings demonstrate that the single ANP32A gene edit is not robust enough for application in the production of chickens, according to the team.

Gene editing

Scientists from University of Edinburgh, Imperial College London and the Pirbright Institute bred the chickens using gene editing techniques to alter the section of DNA responsible for producing the protein ANP32A. During an infection, flu viruses hijack this molecule to help replicate themselves.

When the ANP32A gene-edited chickens were exposed to a normal dose of the H9N2-UDL strain of avian influenza virus – commonly known as bird flu – 9 out of 10 birds remained uninfected and there was no spread to other chickens.

Partial protection

The research team then exposed the gene-edited birds to an artificially high dose of avian influenza virus to further test their resilience.

When exposed to the high dose, half of the group – 5 out of 10 birds – became infected. However, the gene edit did provide some protection, with the amount of virus in the infected gene-edited chickens much lower than the level typically seen during infection in non-gene-edited chickens.

The gene edit also helped to limit onward spread of the virus to just one of four non-gene-edited chickens placed in the same incubator. There was no transmission to gene-edited birds.

Viral evolution

Scientists found that in the ANP32A gene-edited birds, the virus had adapted to enlist the support of two related proteins – ANP32B and ANP32E – to replicate.

Following lab tests, scientists found that some of the mutations enabled the virus to utilise the human version of ANP32, but its replication remained low in cell cultures from the human airway.

Experts say that additional genetic changes would be needed for the virus to infect and spread effectively in humans.

However, the findings demonstrate that the single ANP32A gene edit is not robust enough for application in the production of chickens, according to the team.

Further edits

To prevent the emergence of escape viruses – viruses that adapt to evade the gene edit and cause infection – the research team next targeted additional sections of DNA responsible for producing all three proteins – ANP32A, ANP32B and ANP32E – inside lab-grown chicken cells.

In cell cultures in the lab, growth of the virus was successfully blocked in cells with the three gene edits.

The next step will be to try to develop chickens with edits to all three genes. No birds have been produced yet.

The study highlights the importance of responsible gene editing and the need to be alert to the risks of driving viral evolution in unwanted directions if complete resistance is not achieved, experts say.

Bird flu is a major global threat, with a devastating impact in both farmed and wild bird populations. In the UK alone, the current outbreak of H5N1 bird flu has decimated seabird populations and cost the poultry industry more than £100 million in losses.

In rare instances, mutations in the bird flu virus allow it to infect people and cause serious illness. Efforts to control the spread of the disease are urgently needed.

“Bird flu is a great threat to bird populations. Vaccination against the virus poses a number of challenges, with significant practical and cost issues associated with vaccine deployment. Gene-editing offers a promising route towards permanent disease resistance, which could be passed down through generations, protecting poultry and reducing the risks to humans and wild birds. Our work shows that stopping the spread of avian influenza in chickens will need several simultaneous genetic changes.” Professor Mike McGrew, The study’s principal investigator, from the University of Edinburgh’s Roslin Institute

“This work is an exciting collaboration that fuses our expertise in virology with the world-leading genetic capability at the Roslin Institute. Although we haven’t yet got the perfect combination of gene edits to take this approach into the field, the results have told us a lot about how influenza virus functions inside the infected cell and how to slow its replication.” Professor Wendy Barclay, Imperial College London

The research was funded by UKRI-BBSRC, which also provides strategic funding to The Roslin Institute, and was supported by Edinburgh Innovations, the University’s commercialisation service.

Ryan O’Hare’s October 10, 2023 Imperial College London (ICL) press release offers a slightly different perspective on the same work, Note: A link has been removed,

Scientists have successfully used gene editing techniques to limit the spread of bird flu in chickens.

In a UK first, researchers have been able to restrict, but not completely block, the avian influenza virus from infecting the birds by precisely altering a small section of their DNA.

The modified birds showed no signs that the change had any impact on the animals’ health or well-being.

But the researchers say that while the findings are encouraging, further gene edits would be needed to produce chickens which cannot be infected by bird flu.

The study, carried out by researchers from the University of Edinburgh, Imperial College London and the Pirbright Institute, is published in the journal Nature Communications.

Professor Wendy Barclay, Head of the Department of Infectious Disease at Imperial College London, said: “This work is an exciting collaboration that fuses our expertise in virology with the world world-leading genetic capability at the Roslin Institute.

“Although we haven’t yet got the perfect combination of gene edits to take this approach into the field, the results have told us a lot about how influenza virus functions inside the infected cell and how to slow its replication.”

Global Threat

Bird flu is a major global threat, with a devastating impact in both farmed and wild bird populations. In the UK alone, the current outbreak of H5N1 bird flu has decimated seabird populations and cost the poultry industry more than £100 million in losses.

In the latest study, researchers aimed to test whether precise edits to the chicken’s genome could potentially generate birds which are resistant to the virus.

The team bred chickens with small edits to a gene called ANP32A. During an infection, influenza viruses hijack the ANP32A protein to help replicate themselves.

But when the gene-edited birds were exposed to a normal dose of virus (the H9N2 strain of avian influenza), 9 out of 10 birds remained uninfected and there was no spread to other chickens.

When the birds were exposed to an artificially high dose of virus, only half of them became infected. The single gene edit also provided some protection against transmission, with a much lower amount of virus in infected gene-edited birds compared to non-edited birds.

In addition, the edit also helped to limit onward spread of the virus to just one of four non-edited chickens placed in the same incubator. There was no transmission to gene-edited birds.

Triple edits

Analysis revealed that in the edited birds, the virus adapted to enlist the support of two related proteins to replicate – ANP32B and ANP32E.

Following lab tests, the researchers found some of the mutations may enable the virus to utilise the human version of ANP32, but replication remained low in cell cultures from the human airway. The researchers stress that additional genetic changes would be needed for the virus to have the potential to infect and spread effectively in humans.

According to the team, the findings demonstrate that a single gene edit is not robust enough to produce resistant chickens. To prevent the emergence of viruses able to adapt to the single edit, the team next used a triple edit to target additional proteins (ANP32A, ANP32B and ANP32E) in lab-grown chicken cells.

In cell cultures in the lab, growth of the virus was successfully blocked in cells with edits to all three genes. In future, researchers hope to develop chickens with this triple edit, but no birds have been produced at this stage.

According to the researchers, the study highlights the importance of responsible gene editing and the need to be alert to the risks of driving viral evolution in unwanted directions if complete resistance is not achieved, experts say.

Professor Mike McGrew, from the University of Edinburgh’s Roslin Institute and principal investigator of the study, said: “Bird flu is a great threat to bird populations. Vaccination against the virus poses a number of challenges, with significant practical and cost issues associated with vaccine deployment.

“Gene-editing offers a promising route towards permanent disease resistance, which could be passed down through generations, protecting poultry and reducing the risks to humans and wild birds. Our work shows that stopping the spread of avian influenza in chickens will need several simultaneous genetic changes.”

A non-gene-edited chicken (left) pictured next to an ANP32A gene-edited chicken (right). Image credit: Norrie Russell Courtesy: University of Edinburgh

There’s also an October 10, 2023 article by Jon Cohen for Science.org, which gives some idea of how much work it took to get to this point, Note: Links have been removed,

For 3 decades, Helen Sang has tinkered with the genomes of chickens to try to make the birds resistant to the flu viruses that periodically devastate flocks and raise fears of a human pandemic. Now, as an especially virulent strain of avian influenza sweeps through poultry and wild birds around the world, the geneticist at the University of Edinburgh’s Roslin Institute has her first solid success. Using the CRISPR gene editor and recent findings about what makes poultry vulnerable to flu, Sang and colleagues from three other institutions have created chickens that can resist real-life doses of avian flu viruses. “Sticking to it gets you somewhere in the end,” she says.

The result, published today [October 5, 2023] in Nature Communications, is “a long-awaited achievement,” says Jiří Hejnar, a virologist at the Czech Academy of Sciences’s Institute of Molecular Genetics whose group showed in 2020 that CRISPR-edited chickens could resist a cancer-causing virus. But farmers won’t be raising flu-proof chickens anytime soon. The edited birds still became infected when exposed to larger amounts of the flu virus. And the strategy raises a safety concern: chickens edited this way could, in theory, drive the evolution of flu variants better at infecting people. “What this showed is a proof of concept,” says Wendy Barclay, a virologist at Imperial College London who worked on the new study. “But we’re not there yet.”

Here’s a link to and a citation for the paper,

Creating resistance to avian influenza infection through genome editing of the ANP32 gene family by Alewo Idoko-Akoh, Daniel H. Goldhill, Carol M. Sheppard, Dagmara Bialy, Jessica L. Quantrill, Ksenia Sukhova, Jonathan C. Brown, Samuel Richardson, Ciara Campbell, Lorna Taylor, Adrian Sherman, Salik Nazki, Jason S. Long, Michael A. Skinner, Holly Shelton, Helen M. Sang, Wendy S. Barclay & Mike J. McGrew. Nature Communications volume 14, Article number: 6136 (2023) DOI: https://doi.org/10.1038/s41467-023-41476-3 Published: 10 October 2023

This paper is open access.

CRISPR-like system found in animals

I trust the eukaryotes will not be suing for intellectual property rights. (For anyone who’s interested in CRISPR [clustered regularly interspaced short palindromic repeats) and associated intellectual property (specifically, patent) issues, see my March 15, 2017 posting “CRISPR patent decision: Harvard’s and MIT’s Broad Institute victorious—for now.” It’s not up-to-date but as far as I know there haven’t been any major intellectual property developments since. If I’m wrong, please let me know in the Comments section of this posting.)

A june 28, 2023 news item on phys.org announces research suggesting there are naturally occurring CRISPR-like capabilities in some species,

A team of researchers led by Feng Zhang at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT [Massachusetts Institute of Technology] has uncovered the first programmable RNA-guided system in eukaryotes—organisms that include fungi, plants, and animals.

In a study in Nature, the team describes how the system is based on a protein called Fanzor. They showed that Fanzor proteins use RNA as a guide to target DNA precisely, and that Fanzors can be reprogrammed to edit the genome of human cells. The compact Fanzor systems have the potential to be more easily delivered to cells and tissues as therapeutics than CRISPR/Cas systems, and further refinements to improve their targeting efficiency could make them a valuable new technology for human genome editing

A june 28, 2023 Broad Institute of MIT and Harvard news release by Leah Eisenstadt (also on EurekAlert), which originated the news item, provides more context for the research,

CRISPR/Cas was first discovered in prokaryotes (bacteria and other single-cell organisms that lack nuclei) and scientists including Zhang’s lab have long wondered whether similar systems exist in eukaryotes. The new study demonstrates that RNA-guided DNA-cutting mechanisms are present across all kingdoms of life.

“CRISPR-based systems are widely used and powerful because they can be easily reprogrammed to target different sites in the genome,” said Zhang, senior author on the study and a core institute member at the Broad, an investigator at MIT’s McGovern Institute, the James and Patricia Poitras Professor of Neuroscience at MIT, and a Howard Hughes Medical Institute investigator. “This new system is another way to make precise changes in human cells, complementing the genome editing tools we already have.”

Searching the domains of life

A major aim of the Zhang lab is to develop genetic medicines using systems that can modulate human cells by targeting specific genes and processes. “A number of years ago, we started to ask, ‘What is there beyond CRISPR, and are there other RNA-programmable systems out there in nature?’” said Zhang.

Two years ago, Zhang lab members discovered a class of RNA-programmable systems in prokaryotes called OMEGAs, which are often linked with transposable elements, or “jumping genes”, in bacterial genomes and likely gave rise to CRISPR/Cas systems. That work also highlighted similarities between prokaryotic OMEGA systems and Fanzor proteins in eukaryotes, suggesting that the Fanzor enzymes might also use an RNA-guided mechanism to target and cut DNA.

In the new study, the researchers continued their study of RNA-guided systems by isolating Fanzors from fungi, algae, and amoeba species, in addition to a clam known as the Northern Quahog. Co-first author Makoto Saito of the Zhang lab led the biochemical characterization of the Fanzor proteins, showing that they are DNA-cutting endonuclease enzymes that use nearby non-coding RNAs known as ωRNAs to target particular sites in the genome. It is the first time this mechanism has been found in eukaryotes, such as animals.

Unlike CRISPR proteins, Fanzor enzymes are encoded in the eukaryotic genome within transposable elements and the team’s phylogenetic analysis suggests that the Fanzor genes have migrated from bacteria to eukaryotes through so-called horizontal gene transfer.

“These OMEGA systems are more ancestral to CRISPR and they are among the most abundant proteins on the planet, so it makes sense that they have been able to hop back and forth between prokaryotes and eukaryotes,” said Saito.

To explore Fanzor’s potential as a genome editing tool, the researchers demonstrated that it can generate insertions and deletions at targeted genome sites within human cells. The researchers found the Fanzor system to initially be less efficient at snipping DNA than CRISPR/Cas systems, but by systematic engineering, they introduced a combination of mutations into the protein that increased its activity 10-fold. Additionally, unlike some CRISPR systems and the OMEGA protein TnpB, the team found that a fungal-derived Fanzor protein did not exhibit “collateral activity,” where an RNA-guided enzyme cleaves its DNA target as well as degrading nearby DNA or RNA. The results suggest that Fanzors could potentially be developed as efficient genome editors.

Co-first author Peiyu Xu led an effort to analyze the molecular structure of the Fanzor/ωRNA complex and illustrate how it latches onto DNA to cut it. Fanzor shares structural similarities with its prokaryotic counterpart CRISPR-Cas12 protein, but the interaction between the ωRNA and the catalytic domains of Fanzor is more extensive, suggesting that the ωRNA might play a role in the catalytic reactions. “We are excited about these structural insights for helping us further engineer and optimize Fanzor for improved efficiency and precision as a genome editor,” said Xu.

Like CRISPR-based systems, the Fanzor system can be easily reprogrammed to target specific genome sites, and Zhang said it could one day be developed into a powerful new genome editing technology for research and therapeutic applications. The abundance of RNA-guided endonucleases like Fanzors further expands the number of OMEGA systems known across kingdoms of life and suggests that there are more yet to be found.

“Nature is amazing. There’s so much diversity,” said Zhang. “There are probably more RNA-programmable systems out there, and we’re continuing to explore and will hopefully discover more.”

The paper’s other authors include Guilhem Faure, Samantha Maguire, Soumya Kannan, Han Altae-Tran, Sam Vo, AnAn Desimone, and Rhiannon Macrae.

About Broad Institute of MIT and Harvard
Broad Institute of MIT and Harvard was launched in 2004 to empower this generation of creative scientists to transform medicine. The Broad Institute seeks to describe the molecular components of life and their connections; discover the molecular basis of major human diseases; develop effective new approaches to diagnostics and therapeutics; and disseminate discoveries, tools, methods and data openly to the entire scientific community.

Founded by MIT, Harvard, Harvard-affiliated hospitals, and the visionary Los Angeles philanthropists Eli and Edythe L. Broad, the Broad Institute includes faculty, professional staff and students from throughout the MIT and Harvard biomedical research communities and beyond, with collaborations spanning over a hundred private and public institutions in more than 40 countries worldwide.

About McGovern Institute for Brain Research at MIT
The McGovern Institute is an inclusive and collaborative community of MIT scientists, engineers, and support staff who work together to unravel the mysteries of the brain. Our researchers are committed to meeting two of the greatest challenges of modern science: understanding how the brain works and discovering new ways to prevent or treat brain disorders. To address this scientific challenge, we study the brain at many levels and collaborate with academic, clinical, and industry partners around the world.

The McGovern Institute was established in 2000 by technology entrepreneur Lore Harp McGovern and the late Patrick J. McGovern, former chairman of International Data Group (IDG). Our director is Robert Desimone, the Doris and Don Berkey Professor of Neuroscience at MIT and former head of intramural research at the National Institute of Mental Health. The McGovern Institute has grown from six founding faculty members to more than 20 distinguished investigators including one Nobel laureate and six members of the National Academy of Sciences.

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Here’s a link to and a citation for the paper,

Fanzor is a eukaryotic programmable RNA-guided endonuclease by Makoto Saito, Peiyu Xu, Guilhem Faure, Samantha Maguire, Soumya Kannan, Han Altae-Tran, Sam Vo, AnAn Desimone, Rhiannon K. Macrae & Feng Zhang. Nature (2023) DOI: https://doi.org/10.1038/s41586-023-06356-2 Published: 28 June 2023

This paper is behind a paywall.

Gene-edited food: better tasting and/or allergen-free?

I have two items about gene-edited food. One is from the Canadian Broadcasting Corporation (CBC) and the other is from Hiroshima University (Japan).

Better tasting food?

Cherries without pits do not fit my definition of better tasting food but it’s just one of the touted ‘improvements’.

https://i.cbc.ca/1.6513602.1684353993!/fileImage/httpImage/image.jpg_gen/derivatives/16x9_780/a-little-dirt-never-hurt-01.jpg
Can you imagine eating cherries without having to deal with its pits? That could be a reality thanks to gene-editing tools like CRISPR. (Ben Nelms/CBC)

A May 18, 2023 article by Mouhamad Rachini for CBC’s radio programme, The Current, features information from a radio segment on gene-edited food,

When Michael Wolf tried a new type of mustard green that had been gene-edited to taste less bitter, he came away impressed.

“I don’t necessarily like my food very bitter, so I appreciated it,” Wolf, founder of the food tech publication The Spoon, told The Current’s Matt Galloway.

Food scientists are starting to use gene-editing technology, called CRISPR [clustered regularly interspaced short palindromic repeats], to change certain features of some Canadians’ favourite fruits and vegetables. For example, scientists told Wolf that the technology could be used to create cherries without a pit.

Pairwise, a North Carolina-based gene-editing startup, recently rolled out a mustard green engineered to be less bitter than the original plant. It’s the first CRISPR-edited food to hit the U.S. market. 

Although the gene-edited mustard greens haven’t appeared in Canada yet, the process could find a home here very soon.

Earlier this month, Minister of Agriculture and Agri-Food Marie-Claude Bibeau announced that the Canadian Food Inspection Agency (CFIA) seed guidelines now allow for some modified plants.

The updated rules now allow seeds created through gene-editing without an independent safety assessment by the government, as long as they aren’t spliced with DNA from other types of fruits or vegetables, or altered to make them pesticide-resistant. [emphasis mine]

Wolf explained further that gene-editing with CRISPR has some key differences from other types of genetic modification for food, which has been around for some time.

“[With genetic modification], you’re maybe inserting a foreign DNA into a molecule. But with CRISPR, what it’s essentially doing is just cutting out undesirable traits,” he said. [emphasis mine]

“So you’re not really inserting something that might be foreign to the organism. So it’s something that is a bit, I guess, less concerning for a lot of people who are worried about GMO because that takes away that concern.” [emphasis mine]

“Removing bitterness in a vegetable, I believe, is doing a disservice to our palate,” Dionisia Roman-Osicki of Virden, Man., wrote to The Current. “You can’t be a foodie without recognizing the value of bitterness in food.”

Organic farmer Antony John said there are already “cultural methods” to sweeten the taste and nutritional values of certain foods without genetic modification, such as carrots.

“The cold temperatures causes the carrots to provide an antifreeze, and that antifreeze is sugar,” said John, co-owner of the Soiled Reputation farm in Sebringville, Ont. “So they convert the starch in their roots into sugars. So letting your carrots grow when it’s cold and when there’s subzero temperatures will enhance the sugar in it.”

The radio segment embedded in Rachini’s May 18, 2023 article is 13 mins. 14 secs.

Allergen-free eggs

Over at Hiroshima University, a May 17, 2023 press release (also on EurekAlert but published May 16, 2023) announces research into making eggs safer for people who have allergies, Note 1: The researchers have used a different kind of gene-editing (or genome-editing) technique Note 2: Links have been removed,

Researchers have developed a chicken egg that may be safe for people with egg white allergies. Chicken egg allergies are one of the most common allergies in children. Though most children outgrow this allergy by age 16, some will still have an egg allergy into adulthood. Egg white allergies can cause a variety of symptoms, including vomiting, stomach cramps, breathing problems, hives, and swelling and some people with egg white allergies are unable to receive certain flu vaccines.

Using genome editing technology, researchers have produced an egg without the protein that causes egg white allergies. This protein, called ovomucoid, accounts for approximately 11% of all the protein in egg whites.

Research detailing the food safety profile of this modified egg, called the OVM-knockout, was detailed in a paper published in Food and Chemical Toxicology in April 2023.

“To use OVM-knockout chicken eggs as food, it is important to evaluate its safety as food. In this study, we examined the presence or absence of mutant protein expression, vector sequence insertion, and off-target effects in chickens knocked out with OVM by platinum transcription activator-like effector nucleases (TALENs),” said Ryo Ezaki, an assistant professor at the Graduate School of Integrated Sciences for Life at Hiroshima University in Hiroshima, Japan. TALENs are restriction enzymes that recognize specific DNA sequences and break or cut them.

In order to develop the OVM-knockout eggs, researchers needed to detect and eliminate the ovomucoid protein in the egg whites. TALENs were engineered to target a piece of RNA called exon 1, which codes for specific proteins. The eggs produced from this technique were then tested to ensure there was no ovomucoid protein, mutant ovomucoid protein, or other off-target effects. The eggs had the desired frameshift mutation, which is a mutation created by inserting or deleting nucleotide bases in a gene, and none of them expressed mature ovomucoid proteins. Anti-ovomucoid and anti-mutant ovomucoid antibodies were used to detect any traces of the protein, but there was no evidence of ovomucoid in the eggs. This means that mutant ovomucoids could not create new allergens. This is an important step in determining the safety profile of the eggs.

Other gene editing tools, such as CRISPR, tend to have off-target mutagenesis effects. This means that new mutations are prompted by the gene editing process. However, whole genome sequencing of the altered egg whites showed mutations, which were possibly off-target effects, were not localized to the protein-coding regions.

“The eggs laid by homozygous OVM-knockout hens showed no evident abnormalities. The albumen contained neither the mature OVM nor the OVM-truncated variant,” said Ezaki. “The potential TALEN-induced off-target effects in OVM-knockout chickens were localized in the intergenic and intron regions. Plasmid vectors used for genome editing were only transiently present and did not integrate into the genome of edited chickens. These results indicate the importance of safety evaluations and reveal that the eggs laid by this OVM knockout chicken solve the allergy problem in food and vaccines.”

Looking ahead, researchers will continue to verify the safety profile of the OVM-knockout eggs. Because some people are highly allergic to this specific protein, even small amounts of ovomucoid can cause a reaction. Researchers will need to perform additional immunological and clinical studies to determine the safety of the OVM-knockout eggs. At this time, researchers have determined that OVM-knockout eggs are less allergenic than standard eggs and can be safely used in heat-processed foods that patients with egg allergies can eat. “The next phase of research will be to evaluate the physical properties and processing suitability of OVM-knockout eggs, and to confirm their efficacy through clinical trials,” said Ezaki. “We will continue to conduct further research toward the practical application of allergy-reduced eggs.”

Here’s a link to and a citation for the paper,

Transcription activator-like effector nuclease-mediated deletion safely eliminates the major egg allergen ovomucoid in chickens by Ryo Ezaki, Tetsushi Sakuma, Daisuke Kodama, Ryou Sasahara, Taichi Shiraogawa, Kennosuke Ichikawa, Mei Matsuzaki, Akihiro Handa, Takashi Yamamoto & Hiroyuki Horiuchi. Food and Chemical Toxicology Volume 175, May 2023, 113703 DOI: https://doi.org/10.1016/j.fct.2023.113703

This paper is open access.

A new lipid nanoparticle (LNP) delivery system for CRISPR-Cas9) gene editing

The first time lipid nanoparticles were mentioned here as a delivery system for CRISPR-Cas9 editing was in a January 26, 2018 posting featuring work at the Massachusetts Institute of Technology (MIT). This latest research on the topic comes from Japan according to a March 2, 2023 news item on phys.org,

Gene therapy is a potential mode of treatment for a wide variety of diseases caused by genetic mutations. While it has been an area of diverse and intense research, historically, only a very few patients have been treated using gene therapy—and fewer still cured. The advent of the genetic modification technique called CRISPR-Cas9 in 2012 has revolutionized gene therapy—as well as biology as a whole—and it has recently entered clinical trials for the treatment of some diseases in humans.

Haruno Onuma, Yusuke Sato and Hideyoshi Harashima at Hokkaido University have developed a new delivery system for CRISPR-Cas9, based on lipid nanoparticles (LNPs), that could greatly increases the efficiency of in vivo gene therapy. Their findings were published in the Journal of Controlled Release.

A March 2, 2023 Hokkaido University press release (also on EurekAlert), which originated the news item, provides details about the researchers’ new technique,

“There are broadly two ways of treating diseases with gene therapy,” Sato explained, “ex vivo, where cells are subjected to the desired modifications in the laboratory and then introduced into the patient, and in vivo, where the treatment is administered to the patient to change the cells in their body. Safe and effective in vivo treatment is the ultimate aspiration of gene therapy, as it would be a straightforward process for patients and healthcare providers. LNPs can function as a vehicle for the safe and effective delivery of such therapies.”

CRISPR-Cas9 consists of a large molecule composed of the Cas9 protein and guide RNA. The guide RNA binds to a specific, complementary DNA sequence, and the Cas9 protein cuts that sequence, allowing it to be modified. The guide RNA can be altered to target specific DNA sequences to be modified.

“In a previous study, we discovered that additional DNA molecules, called ssODNs, ensure that the CRISPR-Cas9 molecule is loaded into the LNPs (CRISPR-LNPs),” Harashima elucidated. “In this study,  we again used ssODNs, but they were carefully designed so that they would not inhibit the function of the guide RNA.” 

Using a guide RNA targeting the expression of a protein called transthyretin, they evaluated the effectiveness of the CRISPR-LNPs in mice models. CRISPR-LNPs with ssODNs that dissociated from the guide RNA at room temperature were most effective at reducing serum transthyretin: two consecutive doses, one day apart, reduced it by 80%.

“We have demonstrated the optimal ssODN sequence affinity that ensures the loading and the release of CRISPR-Cas9 at the target location; and that this system can be used to edit cells in vivo,” concluded Onuma. “We will continue to improve the design of ssODNs, as well as to develop optimal lipid formulations to increase the effectiveness of delivery.” 

The image and caption helped me with better understanding the technique described in the press release,

The RNP-ssODN is designed to ensure the CRISPR-Cas9 molecule is encapsulated by the LNP. Once inside the cells, the ssODN dissociates and CRISPR-Cas9 can carry out its effect. (Haruno Onuma, Yusuke Sato, Hideyoshi Harashima. Journal of Controlled Release. February 10, 2023).

Here’s a link to and a citation for the paper,

Lipid nanoparticle-based ribonucleoprotein delivery for in vivo genome editing by Haruno Onuma, Yusuke Sato, Hideyoshi Harashima. Journal of Controlled Release Volume 355, March 2023, Pages 406-416 DOI: https://doi.org/10.1016/j.jconrel.2023.02.008

This paper is behind a paywall.

Gene editing the lungs with nanoparticles

The nanoparticles in question are lipid nanoparticles designed for delivery to the lungs and they are somewhat similar to the ones in some of the COVID-19 vaccines (mRNA vaccines). From a March 30, 2023 news item on Nanowerk,

Engineers at MIT [Massachusetts Institute of Technology] and the University of Massachusetts Medical School have designed a new type of nanoparticle that can be administered to the lungs, where it can deliver messenger RNA encoding useful proteins.

With further development, these particles could offer an inhalable treatment for cystic fibrosis and other diseases of the lung, the researchers say.

“This is the first demonstration of highly efficient delivery of RNA to the lungs in mice. We are hopeful that it can be used to treat or repair a range of genetic diseases, including cystic fibrosis,” says Daniel Anderson, a professor in MIT’s Department of Chemical Engineering and a member of MIT’s Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science (IMES).

In a study of mice, Anderson and his colleagues used the particles to deliver mRNA encoding the machinery needed for CRISPR/Cas9 gene editing. That could open the door to designing therapeutic nanoparticles that can snip out and replace disease-causing genes.

Engineers at MIT and the University of Massachusetts Medical School have designed a new type of nanoparticle that can be administered to the lungs, where it can deliver messenger RNA encoding useful proteins. Credits: Image: iStock, edited by MIT News

A March 30, 2023 MIT news release, also on EurekAlert, which originated the news item, describes the research in more detail,

Targeting the lungs

Messenger RNA holds great potential as a therapeutic for treating a variety of diseases caused by faulty genes. One obstacle to its deployment thus far has been difficulty in delivering it to the right part of the body, without off-target effects. Injected nanoparticles often accumulate in the liver, so several clinical trials evaluating potential mRNA treatments for diseases of the liver are now underway. RNA-based Covid-19 vaccines, which are injected directly into muscle tissue, have also proven effective. In many of those cases, mRNA is encapsulated in a lipid nanoparticle — a fatty sphere that protects mRNA from being broken down prematurely and helps it enter target cells. 

Several years ago, Anderson’s lab set out to design particles that would be better able to transfect the epithelial cells that make up most of the lining of the lungs. In 2019, his lab created nanoparticles that could deliver mRNA encoding a bioluminescent protein to lung cells. Those particles were made from polymers instead of lipids, which made them easier to aerosolize for inhalation into the lungs. However, more work is needed on those particles to increase their potency and maximize their usefulness. 

In their new study, the researchers set out to develop lipid nanoparticles that could target the lungs. The particles are made up of molecules that contain two parts: a positively charged headgroup and a long lipid tail. The positive charge of the headgroup helps the particles to interact with negatively charged mRNA, and it also help mRNA to escape from the cellular structures that engulf the particles once they enter cells. 

The lipid tail structure, meanwhile, helps the particles to pass through the cell membrane. The researchers came up with 10 different chemical structures for the lipid tails, along with 72 different headgroups. By screening different combinations of these structures in mice, the researchers were able to identify those that were most likely to reach the lungs. 

Efficient delivery

In further tests in mice, the researchers showed that they could use the particles to deliver mRNA encoding CRISPR/Cas9 components designed to cut out a stop signal that was genetically encoded into the animals’ lung cells. When that stop signal is removed, a gene for a fluorescent protein turns on. Measuring this fluorescent signal allows the researchers to determine what percentage of the cells successfully expressed the mRNA.

After one dose of mRNA, about 40 percent of lung epithelial cells were transfected, the researchers found. Two doses brought the level to more than 50 percent, and three doses up to 60 percent. The most important targets for treating lung disease are two types of epithelial cells called club cells and ciliated cells, and each of these was transfected at about 15 percent. 

“This means that the cells we were able to edit are really the cells of interest for lung disease,” Li says. “This lipid can enable us to deliver mRNA to the lung much more efficiently than any other delivery system that has been reported so far.”

The new particles also break down quickly, allowing them to be cleared from the lung within a few days and reducing the risk of inflammation. The particles could also be delivered multiple times to the same patient if repeat doses are needed. This gives them an advantage over another approach to delivering mRNA, which uses a modified version of harmless adenoviruses. Those viruses are very effective at delivering RNA but can’t be given repeatedly because they induce an immune response in the host.

To deliver the particles in this study, the researchers used a method called intratracheal instillation, which is often used as a way to model delivery of medication to the lungs. They are now working on making their nanoparticles more stable, so they could be aerosolized and inhaled using a nebulizer. 

The researchers also plan to test the particles to deliver mRNA that could correct the genetic mutation found in the gene that causes cystic fibrosis, in a mouse model of the disease. They also hope to develop treatments for other lung diseases, such as idiopathic pulmonary fibrosis, as well as mRNA vaccines that could be delivered directly to the lungs.

Here’s a link to and a citation from the paper,

Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing by Bowen Li, Rajith Singh Manan, Shun-Qing Liang, Akiva Gordon, Allen Jiang, Andrew Varley, Guangping Gao, Robert Langer, Wen Xue & Daniel Anderson. Nature Biotechnology (2023) DOI: https://doi.org/10.1038/s41587-023-01679-x Published 30 March 2023

This paper is behind a paywall.

Even a ‘good’ gene edit can go wrong

An October 24, 2022 news item on ScienceDaily highlights research into better understanding problems with ‘good’ CRISPR (clustered regularly interspaced short palindromic repeats) gene editing,

A Rice University lab is leading the effort to reveal potential threats to the efficacy and safety of therapies based on CRISPR-Cas9, the Nobel Prize-winning gene editing technique, even when it appears to be working as planned.

Bioengineer Gang Bao of Rice’s George R. Brown School of Engineering and his team point out in a paper published in Science Advances that while off-target edits to DNA have long been a cause for concern, unseen changes that accompany on-target edits also need to be recognized — and quantified.

Bao noted a 2018 Nature Biotechnology paper indicated the presence of large deletions. “That’s when we started looking into what we can do to quantify them, due to CRISPR-Cas9 systems designed for treating sickle cell disease,” he said.

An October 24, 2022 Rice University news release (also on EurekAlert), which originated the news item, details the concerns (Note: Links have been removed),

Bao has been a strong proponent of CRISPR-Cas9 as a tool to treat sickle cell disease, a quest that has brought him and his colleagues ever closer to a cure. Now the researchers fear that large deletions or other undetected changes due to gene editing could persist in stem cells as they divide and differentiate, thus have long-term implications for health.

“We do not have a good understanding of why a few thousand bases of DNA at the Cas9 cut site can go missing and the DNA double-strand breaks can still be rejoined efficiently,” Bao said. “That’s the first question, and we have some hypotheses. The second is, what are the biological consequences? Large deletions (LDs) can reach to nearby genes and disrupt the expression of both the target gene and the nearby genes. It is unclear if LDs could result in the expression of truncated proteins. 

“You could also have proteins that misfold, or proteins with an extra domain because of large insertions,” he said. “All kinds of things could happen, and the cells could die or have abnormal functions.”

His lab developed a procedure that uses single-molecule, real-time (SMRT) sequencing with dual unique molecular identifiers (UMI) to find and quantify unintended LDs along with large insertions and local chromosomal rearrangements that accompany small insertions/deletions (INDELs) at a Cas9 on-target cut site. 

“To quantify large gene modifications, we need to perform long-range PCR, but that could induce artifacts during DNA amplification,” Bao said. “So we used UMIs of 18 bases as a kind of barcode.”

“We add them to the DNA molecules we want to amplify to identify specific DNA molecules as a way to reduce or eliminate artifacts due to long-range PCR,” he said. “We also developed a bioinformatics pipeline to analyze SMRT sequencing data and quantified the LDs and large insertions.”

The Bao lab’s tool, called LongAmp-seq (for long-amplicon sequencing), accurately quantifies both small INDELs and large LDs. Unlike SMRT-seq, which requires the use of a long-read sequencer often only available at a core facility, LongAmp-seq can be performed using a short-read sequencer.

To test the strategy, the lab team led by Rice alumna Julie Park, now an assistant research professor of bioengineering, used Streptococcus pyogenes Cas9 to edit beta-globin (HBB), gamma-globin (HBG) and B-cell lymphoma/leukemia 11A (BCL11A) enhancers in hematopoietic stem and progenitor cells (HSPC) from patients with sickle cell disease, and the PD-1 gene in primary T-cells.  

They found large deletions of up to several thousand bases occurred at high frequency in HSPCs: up to 35.4% in HBB, 14.3% in HBG and 15.2% in BCL11A genes, as well as on the PD-1 (15.2%) gene in T-cells. 

Since two of the specific CRISPR guide RNAs tested by the Bao lab are being used in clinical trials to treat sickle cell disease, he said it’s important to determine the biological consequences of large gene modifications due to Cas9-induced double-strand breaks. 

Bao said the Rice team is currently looking downstream to analyze the consequences of long deletions on messenger RNA, the mediator that carries code for ribosomes to make proteins. “Then we’ll move on to the protein level,” Bao said. “We want to know if these large deletions and insertions persist after the gene-edited HSPCs are transplantation into mice and patients.”  

Co-authors of the study from Rice are graduate students Mingming Cao and Yilei Fu, alumni Yidan Pan and Timothy Davis, research specialist Lavanya Saxena, microscopist/bioinstrumentation specialist Harshavardhan Deshmukh and Todd Treangen, an assistant professor of computer science, and Emory University’s Vivien Sheehan, an associate professor of pediatrics. 

Bao is the department chair and Foyt Family Professor of Bioengineering, a professor of chemistry, materials science and nanoengineering, and mechanical engineering, and a CPRIT Scholar in Cancer Research.

The National Institutes of Health (R01HL152314, OT2HL154977) supported the research.

Here’s a link to and a citation for the latest paper,

Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing by So Hyun Park, Mingming Cao, Yidan Pan, Timothy H. Davis, Lavanya Saxena, Harshavardhan Deshmukh, Yilei Fu, Todd Treangen, Vivien A. Sheehan, and Gang Bao. Science Advances Vol 8, Issue 42 DOI: 10.1126/sciadv.abo7676 First published online: 21 Oct 2022 Published in print: March 3, 2023

This paper is behind a paywall.

A CRISPR (clustered regularly interspaced short palindromic repeats) anniversary

June 2022 was the 10th anniversary of the publication of a study the paved the way for CRISPR-Cas9 gene editing and Sophie Fessl’s June 28, 2022 article for The Scientist offers a brief history (Note: Links have been removed),

Ten years ago, Emmanuelle Charpentier and Jennifer Doudna published the study that paved the way for a new kind of genome editing: the suite of technologies now known as CRISPR. Writing in [the journal] Science, they adapted an RNA-mediated bacterial immune defense into a targeted DNA-altering system. “Our study . . . highlights the potential to exploit the system for RNA-programmable genome editing,” they conclude in the abstract of their paper—a potential that, in the intervening years, transformed the life sciences. 

From gene drives to screens, and diagnostics to therapeutics, CRISPR nucleic acids and the Cas enzymes with which they’re frequently paired have revolutionized how scientists tinker with DNA and RNA. … altering the code of life with CRISPR has been marred by ethical concerns. Perhaps the most prominent example was when Chinese scientist He Jiankui created the first gene edited babies using CRISPR/Cas9 genome editing. Doudna condemned Jiankui’s work, for which he was jailed, as “risky and medically unnecessary” and a “shocking reminder of the scientific and ethical challenges raised by this powerful technology.” 

There’s also the fact that legal battles over who gets to claim ownership of the system’s many applications have persisted almost as long as the technology has been around. Both Doudna and Charpentier’s teams from the University of California, Berkeley, and the University of Vienna and a team led by the Broad Institute’s Feng Zhang claim to be the first to have adapted CRISPR-Cas9 for gene editing in complex cells (eukaryotes). Patent offices in different countries have reached varying decisions, but in the US, the latest rulings say that the Broad Institute of MIT [Massachusetts Institute of Technology] and Harvard retains intellectual property of using CRISPR-Cas9 in eukaryotes, while Emmanuelle Charpentier, the University of California, and the University of Vienna maintain their original patent over using CRISPR-Cas9 for editing in vitro and in prokaryotes. 

Still, despite the controversies, the technique continues to be explored academically and commercially for everything from gene therapy to crop improvement. Here’s a look at seven different ways scientists have utilized CRISPR.

Fessl goes on to give a brief overview of CRISPR and gene drives, genetic screens, diagnostics, including COVID-19 tests, gene therapy, therapeutics, crop and livestock improvement, and basic research.

For anyone interested in the ethical issues (with an in depth look at the Dr. He Jiankui story), I suggest reading either or both Eben Kirksey’s 2020 book, “The Mutant Project; Inside the Global Race to Genetically Modify Humans,”

An anthropologist visits the frontiers of genetics, medicine, and technology to ask: Whose values are guiding gene editing experiments? And what does this new era of scientific inquiry mean for the future of the human species?

“That rare kind of scholarship that is also a page-turner.”
—Britt Wray, author of Rise of the Necrofauna

At a conference in Hong Kong in November 2018, Dr. He Jiankui announced that he had created the first genetically modified babies—twin girls named Lulu and Nana—sending shockwaves around the world. A year later, a Chinese court sentenced Dr. He to three years in prison for “illegal medical practice.”

As scientists elsewhere start to catch up with China’s vast genetic research program, gene editing is fueling an innovation economy that threatens to widen racial and economic inequality. Fundamental questions about science, health, and social justice are at stake: Who gets access to gene editing technologies? As countries loosen regulations around the globe, from the U.S. to Indonesia, can we shape research agendas to promote an ethical and fair society?

Eben Kirksey takes us on a groundbreaking journey to meet the key scientists, lobbyists, and entrepreneurs who are bringing cutting-edge genetic engineering tools like CRISPR—created by Nobel Prize-winning biochemists Jennifer Doudna and Emmanuelle Charpentier—to your local clinic. He also ventures beyond the scientific echo chamber, talking to disabled scholars, doctors, hackers, chronically-ill patients, and activists who have alternative visions of a genetically modified future for humanity.

and/or Kevin Davies’s 2020 book, “Editing Humanity: The CRISPR Revolution and the New Era of Genome Editing,”

One of the world’s leading experts on genetics unravels one of the most important breakthroughs in modern science and medicine. 

If our genes are, to a great extent, our destiny, then what would happen if mankind could engineer and alter the very essence of our DNA coding? Millions might be spared the devastating effects of hereditary disease or the challenges of disability, whether it was the pain of sickle-cell anemia to the ravages of Huntington’s disease.

But this power to “play God” also raises major ethical questions and poses threats for potential misuse. For decades, these questions have lived exclusively in the realm of science fiction, but as Kevin Davies powerfully reveals in his new book, this is all about to change.

Engrossing and page-turning, Editing Humanity takes readers inside the fascinating world of a new gene editing technology called CRISPR, a high-powered genetic toolkit that enables scientists to not only engineer but to edit the DNA of any organism down to the individual building blocks of the genetic code.

Davies introduces readers to arguably the most profound scientific breakthrough of our time. He tracks the scientists on the front lines of its research to the patients whose powerful stories bring the narrative movingly to human scale.

Though the birth of the “CRISPR babies” in China made international news, there is much more to the story of CRISPR than headlines seemingly ripped from science fiction. In Editing Humanity, Davies sheds light on the implications that this new technology can have on our everyday lives and in the lives of generations to come.

Kevin Davies is the executive editor of The CRISPR Journal and the founding editor of Nature Genetics. He holds an MA in biochemistry from the University of Oxford and a PhD in molecular genetics from the University of London. He is the author of Cracking the Genome, The $1,000 Genome, and co-authored a new edition of DNA: The Story of the Genetic Revolution with Nobel Laureate James D. Watson and Andrew Berry. In 2017, Kevin was selected for a Guggenheim Fellowship in science writing.

I’ve read both books and while some of the same ground is covered, the perspectives diverge somewhat. Both authors offer a more nuanced discussion of the issues than was the case in the original reporting about Dr. He’s work.