CRISPR–Cas9

MEGA-CRISPR tool gives a power boost to cancer-fighting cells

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A system that edits RNA rather than DNA can give new life to exhausted CAR T cells

Coloured scanning electron micrograph (SEM) of T lymphocyte cells (smaller round cells) attached to cancer cells.
Engineered cells called CAR T cells (pictured, red; artificially coloured) swarm cancer cells (green). A tool that edits RNA can restore the efficiency of ‘exhausted’ CAR T cells.

The CRISPR–Cas9 gene-editing system excels at altering and disrupting genes. But the changes it makes are permanent, which can be a big problem if the system goes awry. Now, a CRISPR-based system that targets a cell’s short-lived messenger RNA instead of DNA could provide a more precise and reversible way of designing cell therapies — and even help scientists to discover how different genes work together.

RNA gets its turn

Engineered CRISPR systems generally have two main components: a DNA-cutting enzyme, often Cas9, and a piece of ‘guide’ RNA that directs the enzyme to the stretch of DNA to be edited. One of the system’s most promising medical applications has been its potential use in producing chimeric antigen receptor (CAR) T cells. These are made by engineering the immune foot soldiers called T cells to attack specific proteins on the surfaces of tumour cells. But DNA-editing CRISPR systems can pose safety problems and are relatively inefficient in these cells.CRISPR 2.0: a new wave of gene editors heads for clinical trials

Bioengineer Stanley Qi and immunologist Crystal Mackall, both at Stanford University in California, and their colleagues developed an alternative system, called MEGA (multiplexed effector guide arrays). It has CRISPR guide RNA but swaps the DNA-cutting Cas9 for an RNA-cutting alternative called Cas13d. The CRISPR half of the duo directs Cas13d to a target mRNA, which is produced from a DNA template.

“We are not really touching any DNA,” Qi says. This avoids the risk of inducing permanent changes or, worse, cutting DNA in places other than the designated target. The mRNA doesn’t last very long in a cell, so any mistakes will quickly disappear.

Active cells such as T cells produce a constantly changing variety of mRNA molecules, each directing the production of a specific protein. Cas13d cuts the target mRNA, destroying it and preventing it from churning out its specific protein. This has the same effect as turning off the associated gene. MEGA allowed the researchers to create ‘multiplex’ CRISPR–Cas13d systems that can shut down the production of multiple proteins, effectively turning off up to ten genes at a time.

Rejuvenating exhausted cells

The team used the system to address a shortcoming of CAR-T therapy called T-cell exhaustion. If CAR T cells are activated too many times by a chronic infection or a long-term tumour, they become less effective.

To give a jolt to tired T cells, the researchers designed CRISPR systems that target mRNA molecules involved in functions including energy production and sugar metabolism. T cells treated with some MEGA combinations stopped expressing molecular signals of exhaustion and became better at shrinking tumours in mice.The race to supercharge cancer-fighting T cells

Qi, Mackall and their colleagues also created a version of Cas13d that is switched on only when the CAR T cells are treated with the antibiotic trimethoprim. By varying the doses of trimethoprim, the researchers could ‘tune’ mRNA levels up and down, giving the team precise control over when and how molecular pathways were activated, rather than just shutting them down entirely.

“It’s always thrilling to see how the RNA CRISPR toolbox is applied,” says systems biologist Jonathan Gootenberg at the Massachusetts Institute of Technology in Cambridge. The ability to tune the collection of RNA transcripts, he says, will be especially useful for cell therapies.

Joseph Fraietta, an immunologist at the University of Pennsylvania in Philadelphia, agrees. In his own experience with CRISPR, he says, his group can edit only about three genes in CAR T cells at a time before the cells become unhealthy. “This will open more avenues,” he says. But he cautions that the system requires continuously high levels of Cas13d, which might trigger an immune response.

Mackall and Qi say that MEGA’s ability to tune gene expression allows scientists to vary the levels of a wide array of mRNAs at one time, revealing how different amounts of mRNA from various combinations of genes work together to carry out cellular functions.

Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo

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Abstract

Prime editing enables precise installation of genomic substitutions, insertions and deletions in living systems. Efficient in vitro and in vivo delivery of prime editing components, however, remains a challenge. Here we report prime editor engineered virus-like particles (PE-eVLPs) that deliver prime editor proteins, prime editing guide RNAs and nicking single guide RNAs as transient ribonucleoprotein complexes. We systematically engineered v3 and v3b PE-eVLPs with 65- to 170-fold higher editing efficiency in human cells compared to a PE-eVLP construct based on our previously reported base editor eVLP architecture. In two mouse models of genetic blindness, single injections of v3 PE-eVLPs resulted in therapeutically relevant levels of prime editing in the retina, protein expression restoration and partial visual function rescue. Optimized PE-eVLPs support transient in vivo delivery of prime editor ribonucleoproteins, enhancing the potential safety of prime editing by reducing off-target editing and obviating the possibility of oncogenic transgene integration.

Main

Among current genome editing systems that function in both dividing and nondividing mammalian cells in vitro and in vivo, prime editing1 offers unusual versatility by enabling the replacement of a target DNA sequence with virtually any other specified sequence containing up to several hundred inserted, deleted or substituted base pairs2,3,4,5,6,7,8,9,10,11. This versatility makes PE systems particularly promising for the treatment of a broad range of genetic diseases in humans. A prime editor (PE) is an engineered protein consisting of a catalytically impaired programmable nickase domain (such as a Cas9 nickase) fused to an engineered reverse transcriptase (RT) domain. The prime editing guide RNA (pegRNA) specifies the target protospacer sequence and simultaneously encodes the desired edits in the reverse transcription template in the 3′ extension of the pegRNA. The mechanism of prime editing requires three independent nucleic acid hybridization events before editing can take place and does not rely on double-strand DNA breaks or donor DNA templates. As a result of this mechanism, prime editing is inherently resistant to off-target editing or bystander editing, and can proceed with few indel byproducts or other undesired consequences of double-strand DNA breaks1,12,13,14,15,16,17,18,19,20,21.

Fully realizing the potential of prime editing for research or therapeutic applications in mammals requires safe and efficient methods capable of delivering PEs into tissues in vivo. So far, several groups have reported the in vivo delivery of PE via viral delivery methods, including adenoviruses8 and adeno-associated viruses (AAV)8,9,10,11,12,22,23,24,25. Viral delivery methods, however, require that the transgene be encoded directly in the viral gene expression cassette, limiting transgene size. The AAV genome has a cargo gene size limitation of ~4.7 kb (not including inverted terminal repeats)26, requiring large cargoes such as PEs (6.4 kb in gene size for a first-generation PE) to be split into multiple AAVs25, limiting editing efficiency especially at moderate or low vector doses27. Viral delivery methods also pose potential safety risks including increased off-target editing from sustained transgene expression28 and the possibility of unwanted cargo DNA integration into host cell genomes29. Nonviral delivery methods, such as lipid nanoparticles, avoid some of these issues by packaging editors as transiently expressing messenger RNAs (mRNAs). In vivo nonviral targeting of tissues beyond the liver for efficient therapeutic gene editing remains a challenge30,31, however, despite recent advances targeting hematopoietic stem cells32.

Virus-like particles (VLPs) are potentially promising delivery vehicles that in principle offer key benefits of both viral and nonviral delivery methods33. VLPs are formed by spontaneous assembly and budding of retroviral polyproteins that encapsulate cargo molecules from producer cells. VLPs lack a packaged genome but retain the ability to transduce mammalian cells and release cargo34,35. Previous studies explored VLPs for delivering Cas9 nuclease36,37,38,39,40,41,42,43. We recently reported efficient in vivo delivery of adenine base editor (ABE):single guide RNA (sgRNA) ribonucleoproteins (RNPs) with iteratively engineered virus-like particles (eVLPs)44 that overcame specific molecular bottlenecks in cargo packaging, release and localization.

Engineered VLPs offer several advantages over other delivery methods as a candidate for in vivo PE delivery. First, eVLPs are not subject to stringent cargo size limitations, obviating the requirement of splitting PEs into multiple separate vectors. In addition, eVLPs can package RNPs, the most transient form of gene editing agents, thereby reducing frequency of off-target editing by minimizing the exposure duration of the genome to editing agents44,45,46. Since eVLPs lack DNA34,44, they avoid unwanted integration of viral genetic material into the genomes of transduced cells. Finally, eVLPs can be pseudotyped with different glycoproteins, enabling specific targeting of cell types of interest42 with envelope protein engineering efforts.

In this Article, we report the development of a PE-eVLP system that delivers complete PE systems including pegRNAs and nicking sgRNAs (ngRNAs) as RNPs. Simple replacement of base editors (BEs) with PEs in the optimized BE-eVLP system yielded very low functional delivery of prime editing systems (<1% editing efficiency in cultured mammalian cells). Through systematic identification of PE-eVLP delivery bottlenecks and engineering corresponding solutions, we developed third-generation v3 PE-eVLPs that offer a 79-fold improvement in prime editing efficiency compared to v1 PE-eVLPs in mouse Neuro-2A (N2A) cells and a 170-fold improvement in human HEK293T cells. A single subretinal injection of v3 PE-eVLPs demonstrated efficient in vivo prime editing in mouse models, correcting a 4-bp deletion in Mfrp in the rd6 mouse model of retinal degeneration (15% average efficiency) and correcting an Rpe65 substitution to partially rescue visual function in the rd12 model (7.2% average efficiency). Our study establishes PE-eVLPs as a virus-free method for the in vivo delivery of prime editing systems in RNP form.

Discussion

Through extensive engineering of each major component, we developed an all-in-one virus-like particle that delivers PE RNPs into mammalian cells in culture and in vivo. Recent improvements to prime editing systems, including epegRNAs4, the PEmax architecture2 and MMR evasion2, contributed to improved outcomes with PE-eVLPs. Identification of bottlenecks in cargo packaging yielded PE variants that promote delivery by PE-eVLPs, as well as optimized eVLP architectures that facilitate cargo release and cargo localization. Introducing an additional mechanism for guide RNA recruitment addressed guide RNA packaging limitations, and an alternative v3b PE-eVLP system eliminated the need for covalent fusion to the Gag polyprotein. Together, these improvements yielded 170-fold higher average prime editing efficiency compared to v1.1 PE-eVLPs at a benchmark HEK3 test edit in HEK293T cells.

The optimized v3 and v3b PE-eVLPs systems proved efficacious in vivo. Potent prime editing was achieved in the mouse CNS via neonatal ICV injection, marking the first demonstration of CNS editing with transient delivery of a PE RNP. In the mouse retina, a single injection of v3 PE3-eVLPs precisely corrected a pathogenic 4-bp deletion in the rd6 model of retinal degeneration, restoring production of full-length MFRP protein. In the rd12 mouse model of genetic blindness, v3 PE3-eVLPs achieved comparable prime editing levels to a recently reported triple-vector AAV–PE system23, but using a nonviral, single-particle delivery vehicle, resulting in partial rescue of visual function. These findings demonstrate that v3 and v3b PE-eVLPs can achieve prime editing efficiency comparable to that attained using an AAV–PE delivery system, while avoiding drawbacks of viral delivery systems such as prolonged editor expression that increases off-target editing frequencies and the risk of oncogenic DNA integration29,79. To our knowledge, these findings also represent the first use of PE RNPs to achieve phenotypic rescue of an animal model of genetic disease.

While v3 and v3b PE-eVLPs demonstrated therapeutically relevant editing levels, PE-eVLP systems would benefit from the continued engineering effort for the next-generation PEs and improved eVLP systems. Furthermore, tissue-specific envelope protein engineering could expand the scope of PE-eVLP applications to diverse tissues. The possibility that single-dose, transient delivery of PE RNPs by PE-eVLPs may mitigate clinically relevant immunogenicity80 warrants further investigation. Lastly, future optimization in large-scale eVLP production will be necessary to fully realize the therapeutic potential of eVLPs. Nonetheless, the PE-eVLP system reported here offers unique advantages of nonviral, single-particle delivery of PEs in their most transient form as RNPs, presenting safety and target specificity advantages over DNA or mRNA delivery methods.

New killer CRISPR system is unlike any scientists have seen

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It could lead to new cancer treatments, better diagnostics tests, and more.

crispr-Cas12a2
Researchers Chase Beisel and Oleg Dmytrenko. Credit: The Würzburg Helmholtz Institute for RNA-based Infection Research

A unique CRISPR system that destroys infected cells is unlike anything scientists have ever seen before — and it could revolutionize how we use the powerful gene editing technology in the future.

CRISPR 101: While bacteria can infect humans, they can also be infected — a virus can inject its DNA in a bacterial cell and then use the cell like a virus factory, creating more and more copies of itself inside the cell until it bursts.

Just like we have an immune system to protect us against infections, some bacteria have a defense system called CRISPR.

CRISPR lets bacteria collect short DNA sequences from viruses it has seen before. Once it has a new DNA sequence, the CRISPR system creates CRISPR-associated (Cas) proteins, which contain copies of the sequence written in RNA.

There are several different Cas proteins, but the one we know the most about is Cas9. Once created, it will float around a cell, waiting for its target to try to infect the bacteria again.

If that happens, the RNA sequence in the Cas9 protein will bind to the matching sequence in the viral DNA and cut through both sides of its double helix. The sliced-up viral DNA can then be destroyed by other proteins in the bacteria — stopping the infection.

“It’s a phenomenon that elicits audible gasps from fellow scientists.”Ryan Jackson

In 2012, scientists at Lawrence Berkeley National Laboratory published a paper showing that CRISPR could be used for genome editing. It turned out to be wildly successful, and not just in bacteria — if you correctly program CRISPR-Cas9, you can deactivate or remove genes in plants, animals, and even humans.

Since then, researchers have used CRISPR-Cas9 to treat diseases, create healthier crops, and more. They’ve also discovered and utilized CRISPR proteins beyond Cas9, such as Cas13, which cuts single-stranded RNA molecules rather than double-stranded DNA molecules.

What’s new? Now, researchers in the US and Germany have published two papers detailing a new CRISPR protein — Cas12a2 — which they say is more like a genetic Swiss Army Knife than a pair of scissors.

“With this new system … we’re seeing a structure and function unlike anything that’s been observed in CRISPR systems to date,” said researcher Ryan Jackson from Utah State University.

While other CRISPR systems bind to their target sequence, make their cut, and then stop, the researchers learned through a technique called “cryo-electron microscopy” that when Cas12a2 binds to its target, it seems to “activate,” transforming in shape. 

“It’s a change in structure that’s extraordinary to observe — a phenomenon that elicits audible gasps from fellow scientists,” said Jackson.

Once activated, the protein can bind to any genetic material that comes near it, whether its single-stranded RNA, single-stranded DNA, or double-stranded DNA. Cas12a2 then starts shredding the material, making multiple cuts in indiscriminate locations.

Because the genetic material can belong to the bacteria itself, the result can be cellular death. Essentially, CRISPR causes the infected cell to self-destruct — rather than let it become a virus factory.

“It’s poor for that particular cell, but it protects the whole colony of bacteria so that virus doesn’t spread through it,” said Jackson.

If CRISPR-Cas12a2 is programmable, we might be able to use it to kill cells with cancerous mutations.

CRISPR vs. cancer: The newly published papers detail the structure and function of Cas12a2, but more research is needed to determine how we might be able to harness this system for our benefit.

The good news, so far, is that CRISPR-Cas12a2 looks programmable, meaning we might be able to use it to kill certain cells, such as those with cancerous mutations, while leaving healthy cells unharmed.

“If Cas12a2 could be harnessed to identify, target, and destroy cells at the genetic level, the potential therapeutic applications are significant,” said Jackson.

The future of tests: The protein also has potential as a diagnostic tool.

In the past, scientists have programmed CRISPR systems to produce detectable signals when they bind to specific viral sequences — if that signal is seen after adding the CRISPR tech to a sample of saliva, urine, or some other fluid, researchers know it contains the virus.

The problem is the CRISPR systems in those tests destroy the viral sequences after binding to them, which reduces the tests’ sensitivity, but by inserting a mutation into Cas12a2, researchers found that they could get it to only shred single-stranded DNA after activation. 

“This could lead to a really sensitive diagnostic system.”Jack Bravo

That means a test using Cas12a2 could produce a signal when the protein binds to the target sequence of an RNA virus — the type that causes flu, COVID-19, the common cold, and other infections — without destroying the viral RNA that activated it. 

Because that RNA will still be there to activate other CRISPR proteins floating around, the signal will be boosted

“This could lead to a really sensitive diagnostic system, because we’re not depleting what we’re trying to find,” said researcher Jack Bravo from the University of Texas Austin.

“Today, we don’t have new continents to explore … but here in the molecular biosciences, it’s the new frontier.”Ryan Jackson

In theory, such a diagnostic test would be highly accurate, like the PCR tests used for COVID-19 detection, but inexpensive and easy to use — like the less-sensitive but more convenient at-home COVID-19 tests. 

Tests for new RNA viruses could also be easily produced as soon as we know their genomes.

Looking ahead: As exciting as that is, much more research is needed before we can start harnessing Cas12a2’s potential power as a diagnostic or therapeutic tool.

“We’re just scratching the surface, but we believe Cas12a2 could lead to improved and additional CRISPR technologies that will greatly benefit society,” said Jackson.

“Today, we don’t have new continents to explore or other things to discover in that way,” he added, “but here in the molecular biosciences, it’s the new frontier, and it’s a place where you can make really cool discoveries.”

Are we entering golden age of gene therapy?

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Gene Therapy

Gene therapy has experienced a complete renaissance — where will it go in 2024 and beyond?

Eleven years ago, gene therapy — where defective genes are snipped out of DNA and replaced with healthy ones — became a household name. A landmark paper proved that scientists could precisely manipulate DNA in ways previously thought unimaginable using CRISPR-Cas9, an editing tool adapted from the immune system found in some bacteria. Almost overnight, the idea of designer babies, kill-switch mosquitoes, and cancer-off buttons stormed into mainstream imagination.

Since then, gene therapy has experienced a complete renaissance, culminating this past November and early December when medical regulators in the U.K. and U.S. officially approved Casgevy, the first CRISPR-based gene therapy for treating two blood disorders: sickle cell anemia and beta-thalassemia (in the U.S., the new therapy has yet to be approved for the latter).

Casgevy is emblematic of gene therapy’s rapidly shifting expectations and direction. Numerous clinical trials are now underway across the globe, and we will undoubtedly see more and more gene-editing-based treatments making the approval list, changing the lives of countless individuals living with intractable health conditions and diseases.

“The future [of gene therapy] is very bright,” Kevin Davies, the executive editor of The CRISPR Journal and author of Editing Humanity: The CRISPR Revolution and the New Era of Genome Editing, tells Inverse. “But I don’t don’t think anybody in the field wants to get too complacent because it was less than 25 years ago that we were riding a similar initial wave of enthusiasm for the technology.”

It’s true. Long before the Human Genome Project would ever decode a DNA sequence, ambitious scientists were spurred by advances in biotechnology and the early success of initial human trials. In 1990, then-4-year-old Ashanti DeSilva became the first person to be successfully treated with gene therapy. The treatment, a precursor of sorts to CRISPR-based gene therapy, cured her of a rare immune-related genetic disorder.

But those high hopes of tweaking genes to prevent or treat disease were suddenly dashed when, in 1999, a teenager named Jesse Gelsinger, who had a rare metabolic disorder, died within four days of receiving an experimental gene therapy at the University of Pennsylvania. In response, the U.S. Food and Drug Administration (FDA) suspended the university’s entire gene therapy program — which had been the largest in the world at the time — and launched investigations into 69 other gene therapy trials that were underway across the United States. Years later, when CRISPR entered the world, enthusiasm rose again — and here we are.

So what will this new wave — Gene Therapy 2.0, if you will — look like? Certainly, a promising frontier for tackling not only rare but common diseases and exquisitely precise gene-editing tools. With it, though, will come side effects, including exorbitant prices, barriers to access, and a lingering, gigantic cause for concern because once you edit an embryo, there’s no turning back. Here’s what 2024 and beyond has in store for our gene-edited future.

Co-winners of the Nobel Prize in Chemistry 2020 Emmanuelle Charpentier of France and Jennifer A Doud...
Emmanuelle Charpentier and Jennifer A Doudna share the 2020 Nobel Prize in Chemistry for their research into CRISPR.JONATHAN NACKSTRAND/AFP/Getty Images

Better editing

In the early years of genetic therapy, scientists didn’t have many tools at their disposal to fix a gene (or genes) at the heart of a disease, Shoukhrat Mitalipov, director of Oregon Health and Science University’s Center for Embryonic Cell and Gene Therapy, tells Inverse.

If someone’s disease was due to a gene mutation or loss, Mitalipov says the fix was to introduce a synthetic, albeit normal copy of that gene with a virus stripped of its infectiousness but still retaining the ability to add new genetic information into DNA. With this new addition, a cell could then make a functional protein. While these essentially viral Ubers remain part of the gene therapy toolkit, the discovery and development of CRISPR-Cas9 gave scientists a more precise grip on the genetic engineering steering wheel.

CRISPR, or clustered regularly interspaced short palindromic repeats, originate from bacteria and archaea and is used by these microorganisms as an immune defense against viruses called phages. The CRISPR system also includes specialized enzymes called CRISPR-associated proteins (or Cas). Together, they look through and remove any genetic sequences that may have been inserted by a sneaky phage or other invader, keeping the microorganism safe from infection.

In 2012, researchers Jennifer Doudna and Emmanuelle Charpentier published a groundbreaking study detailing a novel CRISPR-Cas9 system they programmed to cut specific sites in isolated DNA. This was done using strands of RNA — a molecule that is like a working copy of DNA, containing the direct instructions for protein-making — guiding CRISPR-Cas9 to a specific genetic sequence.

Krishanu Saha, a bioengineer at the University of Wisconsin–Madison whose lab is working on gene therapies for treating blindness, says the precision allowed by CRISPR-Cas9’s programmability is its singular selling point.

“Traditional gene therapy, which we call gene augmentation, is essentially flooding the cell with extra copies of a normal gene; in some cases, this doesn’t work,” Saha tells Inverse. “We found in a few cases, it’s really important to destroy the mutant copy of the [gene] or fix the underlying mutation and that’s where you have to have the precision of CRISPR to go in and specifically do that.”

CRISPR has a unique drawback, however. When it goes in to patch up the bit of DNA as instructed, it does so by fracturing both strands of the DNA double helix. A cell is left to repair the breaks on its own, ideally using the synthetic DNA offered by the CRISPR-Cas9 system. But because it’s a klutzy repairperson, the cell may also introduce errors such as inserting or deleting DNA.

This is why the focus of the next generation of gene editing tools is to try to minimize, as much as possible, the risk of new mutations, says Mitalipov.

These tools include base editing, where specific base pairs — the building blocks of DNA — are swapped out without requiring a double-stranded break. Base editing was used in a recent gene therapy clinical trial treating individuals with a genetic form of high cholesterol called familial hypercholesterolemia. The gene-editing technique, which is based on CRISPR, was developed in 2016 by Harvard University’s David Liu, considered a founding pioneer of CRISPR.

Another CRISPR-based tool is a leveled-up version of base editing. Called prime editing (also co-invented by Liu), it can swap base pairs in addition to inserting and deleting without double-breaking the DNA helix.

More recently, a gene-editing tool called NICER developed by researchers in Japan is said to create little single-strand nicks that didn’t seem to cause mutations, according to a September 2023 Nature Communications study.

Despite the appeal of finer precision and avoiding inadvertent mutations, Mitalipov and Saha say it’s unlikely the original CRISPR-Cas9 system will be ousted or replaced entirely by these newer gene editors.

“Basically, you would have to look at the specific gene mutation and then decide what would be the best — it could be base or prime editing,” says Mitalipov. “So far, prime editing hasn’t been widely used. There’s only one or two labs [doing research] and nothing commercially available. So, it remains to be seen if [prime editing] is really going to be applicable.”

A laboratory staff works at Genethon, a non-profit gene therapy R&D organization in Evry on May 10, ...
A laboratory staff works at Genethon, a non-profit gene therapy R&D organization in Evry, France. ERIC PIERMONT/AFP/Getty Images

More targets

CRISPR-based gene therapies are being devised to treat all sorts of conditions and disorders, from neurological to autoimmune and cancers. Currently, the only FDA-approved therapy using CRISPR is Casgevy; others on the market, such as Luxturna for people with a rare genetic defect that often leads to blindness and Zolgensma for treating spinal muscular atrophy, use a disabled virus bearing a normal version of the target gene to cells.

There are over 1,500 clinical trials for gene and cell therapies registered with ClinicalTrials.gov, and federal regulators are hoping to approve several more in the coming years, reported FierceBiotech in April 2023.

Currently, the focus is treating disorders or diseases caused by mutations in single genes in somatic cells (the body’s non-reproductive cells). This route makes it easier to identify and target relatively straightforward biological mechanisms than cracking at multiple genes acting in complex and sometimes unpredictable ways with which tinkering may lead to unintended, potentially life-threatening consequences. Understandably, since the late 1990s, there’s been a reasonably high regulatory bar for the research a gene therapy requires to meet FDA approval.

However, that doesn’t mean more genetically complex diseases are off the table. Editing multiple genes is quite possible and regularly done with transgenic animals (as well as plants), says Saha. Attempting this engineering feat for human health will take extensive research to uncover the genetic pathways and interactions involved and figuring out how to safely target all these genes with minimal off-target effects.

In the future, we may see gene therapies used increasingly for common health problems, not only rare genetic diseases, says Mitalipov and Saha. For example, a recent clinical trial in people with familial hypercholesterolemia found that one gene therapy targeting a mutated gene behind the build-up of bad cholesterol slashed cholesterol levels on par with similar-acting pharmaceutical drugs. These findings offer a tantalizing glimpse of an exciting beginning for gene therapy within preventative medicine, promising that someday, a simple edit in your genome may protect you against high cholesterol and blood pressure or any other commonplace ailments.

Therapies for all

There’s another barrier that could ultimately prevent even the safest, most promising gene therapy from seeing the light of clinical day: cost. Luxturna, for example, was reported at a whopping $425,000 per eye back in 2018. It’s a bit of a bargain compared to the average million-dollar price tag for emerging gene therapies such as Casgevy, the gene therapy for sickle-cell anemia/beta-thalassemia. The gene therapy market itself was valued at $1.46 billion in 2020 and is estimated to reach over $5 billion by 2028, according to a report by Polaris Market Research.

“How do we get the pricing down is an outrageously important and unsolvable question,” says Davies of The CRISPR Journal. “Some will point to ‘Well, it’s early days, and as more of these therapies get approved, we start to see competition and prices drop’… but we see when companies get a monopoly on something, they seem more than willing to take advantage of the situation.”

Saha says there’s an active discussion within the scientific community about how to make gene therapy equitable within the low- and middle-income countries that make up the Global South. But how gene therapy accessibility will play out in the coming years is yet to be seen.

“One of the key questions in our analysis is, who’s at the table making these decisions? It’s a fairly easy critique to say that the people in the room are not representative in various ways, perhaps Global North versus Global South, socioeconomic, scientific expertise versus the lay public,” says Saha. “There are important questions about power and democracy and whose knowledge should drive policymaking that needs to be settled. I think the deliberation and the process are as important as the end set of guidelines or policies.”

Then there’s the dreaded ethical prophecy augured by science fiction in films like Gattaca, set in a world where genetic engineering and socioeconomic status go hand in hand. Both before and after the infamous incident involving Chinese scientist He Jiankui creating the world’s first CRISPR-edited human babies with a gene for HIV immunity, there have been strict worldwide regulations on any gene editing research involving embryos.

But scientists like Mitalipov are looking into using CRISPR to potentially adjust an embryo’s risk for disease. His own research at OHSU involves gene-editing germline cells — or reproductive cells that pass on genetic information to the offspring — in what Mitalipov calls “IVF gene therapy.”

He says such a technique could help improve the success of embryo implantation during IVF by creating stronger, more viable embryos. Mitalipov acknowledges, however, that there needs to be a robust regulatory framework in place before we can ever truly consider genetically engineered babies.

“In terms of regulation, we have to focus only on those 10,000 gene defects we know today that cause human disease,” says Mitalipov. “It could be easily mandated that gene therapies in embryos have to be towards severe disease in children.”

But that, and the rest of gene therapy’s optimistically bright future, remains hedged with abundant yet much-warranted caution.

CRISPR: Crispy Fries Your DNA

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Story at-a-glance

  • CRISPR gene-editing technology may have significant unintended consequences to your DNA, including large deletions and complex rearrangements
  • The DNA deletions could end up activating genes that should stay “off,” such as cancer-causing genes, as well as silencing those that should be “on”
  • The deletions detected were at a scale of “thousands of bases,” which is more than previously thought and enough to affect adjacent genes
  • As a result of CRISPR-Cas9, DNA may be rearranged, previously distant DNA sequences may become attached, or unrelated sections could be incorporated into the chromosome

By Dr. Mercola

CRISPR gene-editing technology brought science fiction to life with its ability to cut and paste DNA fragments, potentially eliminating serious inherited diseases. CRISPR-Cas9, in particular, has gotten scientists excited because,1 by modifying an enzyme called Cas9, the gene-editing capabilities are significantly improved. That’s not to say they’re perfect, however, as evidenced by a recent study that showed CRISPR may have significant unintended consequences to your DNA, including large deletions and complex rearrangements.2

Many of the concerns to date regarding CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeat, technology have centered on off-target mutations. The featured study, published in Nature Biotechnology, looked at on-target mutations at the site of the “cuts,” revealing potentially dangerous changes that could increase the risk of chronic diseases like cancer.

Is CRISPR Scrambling DNA?

Researchers at the U.K.’s Wellcome Sanger Institute systematically studied mutations from CRISPR-Cas9 in mouse and human cells, focusing on the gene-editing target site. Large genetic rearrangements were observed, including DNA deletions and insertions, that were spotted near the target site.

They were far enough away, however, that standard tests looking for CRISPR-related DNA damage would miss them. The DNA deletions could end up activating genes that should stay “off,” such as cancer-causing genes, as well as silencing those that should be “on.” One of the study’s authors, professor Allan Bradley, said in a statement:3

“This is the first systematic assessment of unexpected events resulting from CRISPR/Cas9 editing in therapeutically relevant cells, and we found that changes in the DNA have been seriously underestimated before now. It is important that anyone thinking of using this technology for gene therapy proceeds with caution, and looks very carefully to check for possible harmful effects.”

The deletions detected were at a scale of “thousands of bases,” which is more than previously thought and enough to affect adjacent genes. For instance, deletions equivalent to thousands of DNA letters were revealed. “In one case, genomes in about two-thirds of the CRISPR’d cells showed the expected small-scale inadvertent havoc, but 21 percent had DNA deletions of more than 250 bases and up to 6,000 bases long,” Scientific American reported.4

The cells targeted by CRISPR try to “stitch things back together,” according to Bradley, “But it doesn’t really know what bits of DNA lie adjacent to each other.” As a result, the DNA may be rearranged, previously distant DNA sequences may become attached, or unrelated sections could be incorporated into the chromosome.5

Cas9, a bacteria enzyme that acts as the “scissors” in CRISPR, actually remains in the body for a period of hours to weeks. Even after the initial DNA segment had been cut out and a new section “pasted” into the gap to repair it, Cas9 continued to make cuts into the DNA. “[T]he scissors continued to cut the DNA over and over again. They found significant areas near the cut site where DNA had been removed, rearranged or inverted,” The Conversation reported.6

Does This Mean CRISPR Isn’t Safe?

It’s too soon to say what the long-term effects of gene-editing technology will be, and there are many variables to the safety equation. The findings likely only apply to CRISPR-Cas9, which cuts through the DNA’s double strand. Other CRISPR technologies exist that may alter only a single strand or not involve cutting at all, instead swapping DNA letters.

There are also CRISPR systems that target RNA instead of DNA and those that could potentially involve only cells isolated from the body, such as white blood cells, which could then be analyzed for potential mutations before being put back into the body.7

The Nature study did make waves in the industry, though, such that within the first 20 minutes of the results being made public three CRISPR companies lost more than $300 million in value.8

Some companies using CRISPR have said they’re already on the lookout for large and small DNA deletions (including one company using the technology to make pig organs that could be transplanted into humans). One company also claims it hasn’t found large deletions in their work on cells that do not divide often (the Nature study used actively dividing cells).9

The researchers are standing by their findings, however, which the journal took one year to publish. During that time, Bradley says, he was asked to conduct additional experiments and “the results all held up.”10 Past studies have also found unexpected mutations, including one based on a study that used CRISPR-Cas9 to restore sight in blind mice by correcting a genetic mutation.

The researchers sequenced the entire genome of the CRISPR-edited mice to search for mutations. In addition to the intended genetic edit, they found more than 100 additional deletions and insertions along with more than 1,500 single-nucleotide mutations.11 The study was later retracted, however, due to insufficient data and a need for more research to confirm the results.12

CRISPR-Edited Cells Could Cause Cancer

Revealing the many complexities of gene editing, CRISPR-Cas9 also leads to the activation of the p53 gene, which works to either repair the DNA break or kill off the CRISPR-edited cell.13

CRISPR actually has a low efficacy rate for this reason, and CRISPR-edited cells that survive are able to do so because of a dysfunctional p53. The problem is that p53 dysfunction is also linked to cancer (including close to half of ovarian and colorectal cancers and a sizable portion of lung, pancreatic, stomach, breast and liver cancers as well).14

In one recent study, researchers were able to boost average insertion or deletion efficiency to greater than 80 percent, but that was because of a dysfunctional p53 gene,15 which would mean the cells could be predisposed to cancer. The researchers noted, ” … it will be critical to ensure that [CRISPR-edited cells] have a functional p53 before and after engineering.”16

A second study, this one by the Karolinska Institute in Sweden, found similar results and concluded, ” … p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9.”17

Some have suggested that if CRISPR could cure one chronic or terminal disease at the “cost” of an increased cancer risk later,18 it could still be a beneficial technology, but most agree that more work is needed and caution warranted.

A CRISPR clinical trial in people with cancer is already underway in China, and the technology has been used to edit human embryos made from sperm from men carrying inherited disease mutations. The researchers successfully altered the DNA in a way that would eliminate or correct the genes causing the inherited disease.19

If the embryos were implanted into a womb and allowed to grow, the process, which is known as germline engineering, would result in the first genetically modified children — and any engineered changes would be passed on to their own children. A February 2017 report issued by the U.S. National Academies of Sciences (NAS) basically set the stage for allowing research on germline modification (such as embryos, eggs and sperm) and CRISPR, but only for the purpose of eliminating serious diseases.

In the U.S., a first of its kind human trial involving CRISPR is currently recruiting participants with certain types of cancer. The trial is going to attempt to use CRISPR to modify immune cells to make them attack tumor cells more effectively. As far as risks from potential mutations, it’s anyone’s guess, but lead researcher Dr. Edward Stadtmauer of the University of Pennsylvania told Scientific American, “We are doing extensive testing of the final cellular product as well as the cells within the patient.”20

Are ‘Designer Babies’ Next?

It’s easy to argue for the merits of CRISPR when you put it in the context of curing deafness, inherited diseases or cancer, and at least 17 clinical trials using gene-editing technologies to tackle everything from gastrointestinal cancer to tumors of the central nervous system to sickle cell disease have been registered in the U.S.21 Another use of the technology entirely is the creation of “designer babies” with a certain eye color or increased intelligence.

About 40 countries have already banned the genetic engineering of human embryos and 15 of 22 European countries prohibit germ line modification.22 In the U.S., the NAS report specifically said research into CRISPR and germline modification could not be for “enhancing traits or abilities beyond ordinary health.” Still, using gene editing to create designer babies is a question of when, not if, with some experts saying it could occur in a matter of decades.23

There are both safety and ethical considerations to think about. With some proponents saying it would be unethical not to use the technology. For instance, Julian Savulescu, an ethicist at the University of Oxford, told Science News he believes parents would be morally obligated to use gene-editing technology to keep their children healthy.

“If CRISPR could … improve impulse control and give a child a greater range of opportunities, then I’d have to say we have the same moral obligation to use CRISPR as we do to provide education, to provide an adequate diet …”24 Others have suggested CRISPR could represent a new form of eugenics, especially since it can only be done via in vitro fertilization (IVF), putting it out of reach of many people financially and potentially expanding inequality gaps.

On the other hand, some argue that countries with national health care could provide free coverage for gene editing, possibly helping to reduce inequalities.25 It’s questions like these that make determining the safety of CRISPR and other gene-editing technology more important now than ever before.

What Does a CRISPR-Enabled Future Hold?

We’ve already entered the era of genetic engineering and CRISPR represents just one piece of the puzzle. It’s an exciting time that could lead to major advances in diseases such as sickle-cell anemia, certain forms of blindness, muscular dystrophy, HIV and cancer, but also one that brings the potential for serious harm. In addition to work in human and animal cells, gene-edited crops, in which DNA is tweaked or snipped out at a precise location, have already been created — and eaten.

To date, the technology has been used to produce soybeans with altered fatty acid profiles, potatoes that take longer to turn brown and potatoes that remain fresher longer and do not produce carcinogens when fried. The latter could be sold as early as 2019.

The gene-editing science, in both plants and animals, is progressing far faster than long-term effects can be fully realized or understood. There are many opportunities for advancement to be had, but they must come with the understanding that unintended mutations with potentially irreversible effects could be part of the package.

Watch the video. URL:https://youtu.be/faSoxyiAAPE

The Progress and Promise of Gene Editing

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Earlier this year, a report prepared for the National Academies urged caution in developing the gene-editing technology known as CRISPR-Cas9, but stopped short of calling for an outright ban. Click here to read MedPage Today’s original report on the Academies’ position. In this follow-up, we review further developments with CRISPR and its regulation.

New technologies such as the CRISPR-Cas9 offer the possibility of altering an individual’s genome, or even a generation’s genome.

Jennifer Doudna, PhD, a geneticist and professor at the University of California Berkeley and the Howard Hughes Medical Institute, created CRISPR in collaboration with Emmanuelle Charpentier, PhD, of Umea University in Sweden, in 2012.

Out of fear the technology could be misused, Doudna advocated a worldwide moratorium on gene editing that involved heritable changes.

Thus far, no researchers have publicly stated that they have made germline alterations in a human embryo with the intent of nurturing it to birth. But over the past year they have inched closer.

In August, researchers at Oregon Health & Science University in Portland, Oregon, led by biologist Shoukhrat Mitalipov, PhD, for the first time in the U.S. demonstrated the potential to edit human embryo DNA to prevent a congenital heart condition known as hypertrophic cardiomyopathy, which may cause heart failure or sudden death.

Then in October, the New Scientist reported that the CRISPR method was showing promise across a range of diseases in animal studies, including in muscular dystrophy and liver disease.

 Most of the research involved ex vivoexperiments — removing cells, editing them in a lab and then replacing them.

While this process is “relatively easy” for immune cells or blood stem cells, “this isn’t possible with most bodily tissues,” noted the New Scientist’s Michael LePage.

Matthew Porteus, MD, PhD, associate professor of pediatrics at Stanford University and an NAM committee member, told a Senate, Health Education, Labor and Pensions Committee in November that the best approach for other conditions such as congenital blindness and muscular dystrophy likely involves in vivo gene editing.

Regarding other conditions studied through ex vivo experiments, Porteus said his lab developed a method for correcting mutations of sickle cells in patients’ stem cells. If a cure is found, it might take only a few “tweaks” to then find a cure for other illnesses, such as severe combined immunodeficiency, he noted.

He anticipates seeing multiple CRISPR-Cas9 clinical trials in the U.S. or Europe in the next 12-18 months, Porteus added.

What’s Next for Gene Editing?

Asked about the most notable breakthroughs in the field right now, R. Alta Charo, JD, co-chair of the Committee on Human Gene Editing and a professor at the University of Wisconsin in Madison, spoke of “the developing capacity to do epigenetic editing,” speaking on her own behalf, in an email to MedPage Today.

“[I]t offers the prospect of making beneficial changes that, because they are reversible, in many cases will pose fewer risks,” she said.

As another benefit, this form of gene editing could be used to respond to conditions that stem from a “constellation of genetic factors” rather than a single mutation, reported Wired.

Researchers have already begun testing epigenetic editing in mice for diseases such as diabetes, acute kidney disease, and muscular dystrophy, Wired noted.

“Successful somatic gene therapy” and the OHSU study “pending confirmation by the scientific community” are the most notable breakthroughs of the year, said Jeffrey Kahn, PhD, MPH, director of the Johns Hopkins Berman Institute of Bioethics in Baltimore, and a member of the NAM committee, in an email to MedPage Today.

However, he noted that pre-implantation genetic diagnosis could have replaced gene editing in the OHSU study. In other words, the researchers ignored one of the NAM committee’s key criteria for heritable gene editing: lack of a reasonable alternative.

Others disagreed.

Because the study did not involve a pregnancy or birth “it constitutes purely laboratory research” and would be “permissible” under committee guidelines, said Charo.

“An emerging area in gene editing is harnessing these new precision engineering tools to edit regions of the genome outside of genes,” said Neville Sanjana, PhD, a core faculty member at the New York Genome Center and a professor at New York University, in an email, responding to the same question.

The ‘Dark Genome’ Emerges as Target

Gene editing tools can help to translate these regulatory and noncoding variants, those outside of the genes — less than 2% are actually in the genes themselves.

This area is sometimes referred to as the “dark genome.”

“Most of our genome is actually this ‘noncoding’ DNA and not in genes (less than 2% is in genes). We understand very little about how this noncoding DNA works and how changes in the sequence (primary sequence — not epigenome) results in changes in gene expression and disease,” said Sanjana.

Sanjana also highlighted the approval of “a multitude” of new gene therapies by the FDA for conditions such as congenital blindness, spinal muscular atrophy and different hemophilias, which he said has also generated a lot of excitement.

Although some gene therapies have been around since the 1990s, not all involve gene editing. However, the approvals represent progress, he noted.

“It is clear that this new modality of therapy — adding back a missing or damaged gene — will open new avenues of medicine,” said Sanjana.

“[I]t is a matter of time before gene editing tools are also part of the gene therapy arsenal to aid in curing disease for which we currently have no therapies,” he added.

As always, oversight will remain important to this process.

“The challenge is to find the ‘just right’ regulatory approach for what are new, emerging, and controversial biotechnologies such as gene editing tools. That often requires some tweaking to get right, and I hope that there is willingness to engage in the discussion necessary to find the appropriate balance of control with a path for innovation,” wrote Kahn.

First Commercialized GMO Maize Was Toxic to Farm Animals

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Genetically engineered (GE) food comes from crops in which genes from one species have been integrated into another species — even between species in which this is biologically impossible in nature. The end result is a gene sequence that would never have occurred naturally.

There are two main types of GE crops:

  1. Herbicide-tolerant crops: Plants engineered to withstand heavy herbicide spraying without sustaining damage, such as Roundup Ready crops
  2. Pesticide-producing crops: So-called Bt plants are engineered to produce their own internal pesticide, so that when a bug takes a bite of the plant, it dies

Trying to control genetic changes via artificial modification is a dangerous game. An organism’s genome is not static but fluid, and its biological functions are interconnected with its environment and vice versa.

Contrary to what the industry would like you to believe, the process of genetic engineering is imprecise at best, and is riddled with unintended and often unforeseeable consequences.

Of course that is about to change with the new technology, CRISPR-Cas9 that I reviewed last month. Once this technology is implemented, we will need to pay very careful attention to what the researchers are planning.

Genetic Engineering Is Riddled With Unintended Consequences

Viruses are typically used to genetically engineer the genes into a new species. These are known as viral transgenes, and there’s a profound lack of understanding of how this process actually works and what the ultimate ramifications are.

Compared to natural genetic modification (vertical gene transfer), artificial genetic modification is inherently hazardous because it lacks the precision of the natural process, enabling genes to be transferred between species that would never have been otherwise exchanged.

Artificial genetic modification uses horizontal gene transfer, which involves injecting a gene from one species into a completely different and naturally incompatible species, yielding unexpected and often unpredictable results — some of which may pose a hazard to animal and human health.

Approval Does Not Mean GE Crops Have a Proven Safety Record

In 1995, Novartis (which later became Syngenta) received approval to cultivate the GE maize known as Bt1761 in the U.S. It was the first Bt corn commercialized for animal feed. Due to controversies, it never gained much market success and the registration was allowed to lapse in 2001.2

In Europe, it was officially withdrawn from the market in 2007. Last month, Professor Gilles-Eric Séralini published a feeding study on this particular Bt corn, showing it was in fact toxic to cows over the long-term.

Prior to its introduction, Novartis had conducted just one feeding test on four cows for the duration of two weeks.3 One of the animals died one week into the test with electrolyte and mucosal problems. No scientific explanation could be found for the death, and the cow was removed from the protocol.

It’s really important to realize that animal feeding trials are not required to be done prior to the commercial release of a GMO, and if they are done, they’re typically extremely small, and very short in duration, like this one was.

Long-Term Studies Keep Finding Serious Health Problems With GMOs and Associated Chemicals

As an expert for the French government within the Biomolecular Engineering Commission, Séralini had access to the industry dossier on Bt176, and expressed strong objections to and concern over the lack of long-term feeding tests — the kind that have since become Séralini’s own hallmark specialty.

As you may recall, Séralini produced the first-ever lifetime feeding study on rats in 2012. The 2-year-long study evaluated lifelong effects of a Monsanto-produced GE corn that is prevalent in the U.S. food supply.

The rats developed massive breast tumors, kidney and liver damage, and early death. The major onslaught of diseases set in during the 13th month, which in human terms equate to about the age of 43, assuming that the average person lives to the age of 80.

Séralini has also investigated the health effects of glyphosate and Roundup.

In a study4,5 published last year, he found that long-term exposure to ultra-low amounts of Roundup — which is used on both GE and conventional crops in ample amounts — may cause tumors, along with liver and kidney damage in rats.

First GE Corn Shown to Be Toxic to Cows in the Long Term

In 1997, Gottfried Glöckner, an award-winning dairy farmer in Germany, became the first farmer to grow and feed Bt176 corn to his prized Holstein cows. The test continued until 2002.

According to Séralini, this was the longest running and most detailed observation of farm animals ever performed for a GE crop.

Since 1986, when Glöckner took over the farm, he’d had no cases of serious disease on his farm. That all changed once he started feeding his cows Bt176 in 1997. As noted on Séralini’s website:6

“When partial paralysis (paresis) accompanied by great fatigue, and problems in the kidneys and mucosal membranes arose in the animals, followed by death in 10 percent of cases, microbial causes were sought. All kinds of analyses were conducted …

At this time, the dose of GMO Bt maize, which had been progressively introduced, had reached 40 percent of the diet. By 2002, the farmer had become convinced that Bt maize was the cause of the diseases. He sued Syngenta and had partial compensation for his losses7 …

After all these court cases ended, Prof. Séralini gained access to veterinary records and to very complete archived data for each cow … For the first time ever, an analysis of these data has been published8 … New scientific data on Bt toxins and a thorough study of the records show that this GMO Bt maize is most probably toxic over the long term.

This study reveals once again the urgent need for specific labeling of the identity and quantity of GMOs, especially in food and feed. Long-term testing of GM food and the pesticides they are designed to contain must be carried out and made public. This is now more essential than ever.”

The Higher the GMO Content, the Greater the Health Risks

As Glöckner increased the amount of Bt176 corn in the cows’ feed, gradually going from 2 to 40 percent over the course of two years, the worse his cows fared. At the outset, 70 percent of his cows produced high yields of milk, which is considered normal.

Once the GMO content of the feed reached 40 percent, a mere 40 percent of his cows were high-yielding. In 2000, milk tested positive for the Bt176 DNA specific fragment, which under European law meant the milk had to be labeled as coming from GE-fed animals.

Peak mortality was reached in 2002, when 10 percent of his cows died after suffering a long period of partial paralysis. Thirty percent of the herd was sick with a variety of ailments.

A number of cows were diagnosed with liver disease, mucosa problems, irregular heart function, mammary gland breaks (which is exactly as disturbing as it sounds: the study includes pictures), and general “abnormal behavior” suggesting chronic lack of energy.

As the GMO ratio peaked, fertility also began to drop significantly. Some of the animals tested positive for Chlamydia, but had no visible infection. Overall, kidney function appeared to be the most affected.

Because the farmer introduced new cows to his herd here and there to replace those who had died or were too sick to be milked, the toxic effects may actually be underestimated, as the replacement animals had not previously eaten the GMO feed, and were therefore exposed to it for a much shorter duration.

Indeed, Séralini points out that toxic effects such as these would likely be missed under common conditions on factory farms with high and rapid animal turnover for that very reason. Especially when the feed is not specifically labeled, identifying the type of GMO and precise amount.

Pesticide-Producing Plants May Also Harm Human Health

Like other Bt crops, Bt176 was genetically engineered to produce Bacillus thuringiensis(Bt toxin) — a pesticide that breaks open the stomach of certain insects and kills them. Bt plants are engineered to produce this pesticide internally, so it’s present in every cell of the plant, from root to tip, and cannot be washed off.

Previous in vitro experiments9,10 have shown that the Bt toxin these plants produce affects human cells, both alone and in combination with glyphosate-based herbicide residues.

Pesticidal crystal proteins Cry1Ab and Cry1Ac, two subspecies of the Bt toxin, were tested on cells from the embryonic kidney cell line 293, looking at specific biomarkers indicating cell death. Concentrations ranged from 10 parts per billion (ppb) up to 100 parts per million (ppm).

Cry1Ab caused cell death starting at 100 ppm. Roundup alone was found to cause necrosis (cell death resulting from acute injury) and apoptosis (cellular “suicide” or self-destruction) starting at 50 ppm, which the researchers noted is “far below agricultural dilutions.”

According to the authors: “In these results, we argue that modified Bt toxins are not inert on nontarget human cells, and that they can present combined side effects with other residues of pesticides specific to GM plants.”

Monsanto and the U.S. Environmental Protection Agency (EPA) claimed the Bt toxin produced inside the plant would be completely safe for human consumption because it would be destroyed in the human digestive system. This has been proven false more than once.

Research11 published in 2007 found that antibiotic resistance marker genes from Bt176 maize were able to survive for longer periods in gastric juices taken from patients on anti-acid drug treatment, thereby potentially increasing the risk of antibiotic resistance. According to the authors:

“Our data indicate the possibility that in particular cases the survival time could be so delayed that, as a consequence, some traits of DNA could reach the intestine. In general, this aspect must be considered for vulnerable consumers (people suffering from gastrointestinal diseases related to altered digestive functionality, physiological problems or drug side-effects) in the risk analysis usually referred to healthy subjects.”

Then, in 2011, doctors at Sherbrooke University Hospital in Quebec found Bt-toxin in the blood of 93 percent of pregnant women tested, 80 percent of umbilical blood in their babies, and 67 percent of non-pregnant women.12 It’s quite clear that Bt toxin is not destroyed when passing through your digestive system, and that it can bioaccumulate in your body.

According to one study,13 Bt toxin may produce a wide variety of immune responses, including elevated IgE and IgG antibodies, typically associated with allergies and infections, and an increase in cytokines, associated with allergic and inflammatory responses — conditions that have markedly risen in prevalence since the advent of Bt crops.

Transgenic Bt Crops Promote Resistant Pests and Destroys Soil Biology

One of the selling points and touted benefits of GE crops like Bt cotton and Bt corn is reduced pesticide usage, as the plant itself will kill any bug that chews on it. As with so many other GMO claims, this one cannot stand up to scrutiny. For starters, just like exaggerated herbicide use has led to the rapid development of resistant superweeds, so have Bt plants led to the emergence of resistant pests.

According to The Times of India,14 farmers in Punjab and Haryana are seeing significant losses of their Bt cotton crops to the whitefly. To address the problem, increasing amounts of pesticides have been applied. During an outbreak in 2002 farmers applied so much pesticide to fend off the whiteflies that soil and groundwater are thought to have been affected.

Many now blame the exaggerated use of pesticides on the clustering of cancer cases being detected among those living in India’s cotton belt. Research15 has also shown that Bt crops, just like topical pesticides and herbicides, alter and destroys soil microbiology. According to the authors:

“Our data showed that the cultivation of Bt maize significantly increased the saturated to unsaturated lipid ratios in soils which appeared to negatively affect microbial activity.” 

Beware: Bt Toxin Produced by Bt Plants Is Not Counted Toward Total Pesticide Exposure

Last but not least, it’s well worth noting that the Bt toxin produced in these Bt crops are NOT included as part of the total human pesticide exposure. This despite the fact that Bt plants are actually registered with the EPA as a pesticide.16 This also helps explain why Bt plants damage the soil just like topical pesticides do.

Ignoring Bt toxin produced by Bt plants, as if it never were to reach a dinner plate, is a gross misrepresentation of facts and outright fraudulent propaganda. How can they claim reductions in pesticide exposure as a result of Bt plants when every single cell of the plant contains it?

And how can they not include the plants in the pesticide usage data when the plant itself is registered as a pesticide? The failure to count the toxin inside the plant, and only counting the pesticides applied topically, is a significant loophole that makes Bt plants appear to provide a benefit that in reality simply isn’t true.

In reality, Bt exposure has likely increased exponentially with the introduction of Bt plants. Why? Because the plant-produced version of the poison is thousands of times more concentrated than the topical spray, and while topically applied Bt toxin biodegrades in sunlight and can be washed off, the Bt toxin in these GE plants does not degrade, nor can it be removed or cleaned off the food since it’s integrated into every cell of the plant.

Besides that, Bt toxin in GE soy, cotton, and corn has also been exempted from residue tolerance levels by the EPA, so absolutely no one is looking for or paying any attention to the amount of Bt toxin you’re exposed to via the food you eat!

How to Avoid Bt Crops

So, if you want to avoid eating Bt plants, which foods end up on the “buy certified organic” list? The following list shows which Bt crops have received approval for commercialization in which countries as of 2013.17,18 (A Bt poplar tree has also been approved for planting in China.)

Cotton is of course not a food, but is used for cotton clothing. The genetic engineering of cotton is one reason why I recommend buying clothing made with organic cotton.

Bt crop Country
Cotton Argentina, Australia, Brazil, Burkina Faso, Canada, China, Colombia, Costa Rica, European Union (EU), India, Japan, Mexico, Myanmar, New Zealand, Pakistan, Paraguay, Philippines, Singapore, South Africa, South Korea, and United States of America (USA)
Eggplant Bangladesh
Maize/Corn Argentina, Australia, Brazil, Canada, Chile, China, Colombia, Egypt, El Salvador, EU, Honduras, Indonesia, Japan, Malaysia, Mexico, New Zealand, Panama, Paraguay, Philippines, Russian Federation, Singapore, South Africa, South Korea, Switzerland, Taiwan, Thailand, Turkey, USA, and Uruguay
Potato (“Atlantic NewLeaf potato”19,20) Australia, Canada, Japan, Mexico, New Zealand, Philippines, Russian Federation, South Korea, and USA
Rice China and Iran
Soybean Argentina, Australia, Brazil,21 Canada, China, Colombia, EU, Japan, Mexico, New Zealand, Paraguay, South Korea, Taiwan, Thailand, USA, Uruguay
Tomato22,23,24 Canada, Chile, and USA

Watch the whistle blowing video by Dr Vandana Shiva. URL:

Source:mercola.com

CRISPR gene editing tech brings countless opportunities and challenges.

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Dr. Thomas Doetschman, Ph.D., examines the embryonic cells used to study and implant mutated and disease genes; if the mutated gene successfully imbeds itself into a sperm or egg cell, the resulting rat that is born will be studied to research the effects of that same disease genes in humans. CRISPR CAS9 is technology that allows the splicing of genes to both remove and replace particular DNA strands. CRISPR can affect either just the patient or his descendants as well, depending on the technique used.

A new genome editing technology known as CRISPR has the potential to revolutionize the way scientists study diseases and genetics.

“I think it’s a really useful tool for science, in fact it’s sort of revolutionizing the speed at which we can accomplish certain things in the laboratory and it has tremendous potential for therapeutic applications,” said Kimberly McDermott, a research associate professor of medicine and an associate professor of cellular and molecular medicine, cancer biology and genetics.

Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR-Cas9, is based off a bacterial immune system, said Thomas Doetschman, professor of cancer biology, genetics and cellular and molecular medicine.

When bacteria become infected by a virus, they take pieces of the virus’s DNA and incorporate it into their own genome. This allows the bacteria to recognize and attack the virus if it ever appears again. This system allows them to destroy the virus, but it also allows them to destroy DNA, Doetschman said.

In developing CRISPR, scientists took a hint from the bacteria.

“What it [CRISPR] actually does is causes a mutation at that site, in the DNA, and then repairs it,” Doetschman said. “And you can repair it in different ways, such that you can actually modify the sequence of the DNA.”

This has enormous implications for the study of genetics and combating human diseases. And while it may sound exciting, human gene editing isn’t all fun and games.

There are two ways the CRISPR technology can be used in humans, Doetschman said. The first way is to alter somatic cells, which don’t get passed down to the next generation. This would only affect the patient who is receiving the treatment. The second way, known as the germline, can have serious long-lasting effects. Altering genes in the germline can produce permanent changes in the patient that will then be passed on to their children.

“There’s two completely different ways of doing this, and the real concern, the big concern, is that it be used by some unscrupulous people to try to change the germline of people, so that you can create progeny that will all have this kind of modification,” Doetschman said.

CRISPR isn’t just for humans; it can be used to edit plant cells as well.

“It could alter genes in a plant so that the plant either becomes resistant to or susceptible to agents that might otherwise kill the plant,” Doetschman said. This could mean disease-resistant plants or increased nutritional content.

One of CRISPR’s greatest contributions is in the realm of research, specifically for understanding normal development and disease processes, McDermott said.

For example, in the future scientists may be able to grow human organs from the patient’s own cells, using CRISPR.

Recent studies on mice and rats have introduced the possibility of using a model organism, such as a pig, to grow human organs, McDermott said.

Another exciting possibility available through CRISPR involves induced pluripotent stem cells, Doetschman said. This process essentially works as a time machine for your cells.

Doetschman describes it as the ability to put your own cells, such as skin cells, in culture and de-differentiate those cells back down to the pluripotent “master key” stem cell, using CRISPR. Once your adult cells are transformed into stem cells, you can make the genetic modifications you’d like, such as correcting a mutation, and then re-differentiate the cells back into the cell type of the tissue you want to correct.

These cells could potentially be engrafted  back into the patient’s disease tissue, Doetschman said.

Dr. Thomas Doetschman, Ph.D., describes a few of the many functions performed in the workspace pictured, which can effectively seal itself to create a sterile and airtight environment in which researchers can operate. CRISPR technology may redefine the future of genetics.

When it comes to working with human therapeutics, safety and regulations are extremely important, McDermott said.

As scientists, their primary concern is to minimize and prevent harm in every way possible. One of these regulations is a patent that was recently issued to the MIT and Harvard-affiliated Broad Institute, one of the centers responsible for creating CRISPR technology.

Despite heavy public controversy surrounding the patent, Doetschman said the patent is a good thing, because it will allow scientists to ensure that CRISPR research is carried out in a safe way, especially in regards to human use.

“I think from a scientist’s perspective, the thing that we’re really focusing on is trying to listen to our colleagues but also the public in general about what are the fears of this technology,” McDermott said. “Of course when you start to edit genes and mutate genes there’s a lot of concerns about what might happen.”

As for the future of human genetics research, both Doetschman and McDermott remain optimistic. CRISPR improves both the efficiency and the accuracy of genome research.

McDermott said while scientists may have had the ability to make mutations in cells in the past, the results were usually inefficient and could produce off-target effects.

CRISPR might not be the cure to every disease, but it is the key to unlock many avenues of research, Doetschman said.

“In terms of the research end of science and medical research, it’s expanding tremendously the scientist’s ability to ask questions about genetic disease,” Doetschman said.

What is CRISPR-Cas9?

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CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.

What is CRISPR-Cas9?

  • CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit parts of the genome? by removing, adding or altering sections of the DNA? sequence.
  • It is currently the simplest, most versatile and precise method of genetic manipulation and is therefore causing a buzz in the science world.

How does it work?

  • The CRISPR-Cas9 system consists of two key molecules that introduce a change (mutation?) into the DNA. These are:
    • an enzyme? called Cas9. This acts as a pair of ‘molecular scissors’ that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed.
    • a piece of RNA? called  guide RNA (gRNA). This consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome.
  • The guide RNA is designed to find and bind to a specific sequence in the DNA. The guide RNA has RNA bases? that are complementary? to those of the target DNA sequence in the genome. This means that, at least in theory, the guide RNA will only bind to the target sequence and no other regions of the genome.
  • The Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut across both strands of the DNA.
  • At this stage the cell? recognises that the DNA is damaged and tries to repair it.
  • Scientists can use the DNA repair machinery to introduce changes to one or more genes? in the genome of a cell of interest.

Diagram showing how the CRISPR-Cas9 editing tool works.

How was it developed?

  • Some bacteria? have a similar, built-in, gene editing system to the CRISPR-Cas9 system that they use to respond to invading pathogens? like viruses,? much like an immune system.
  • Using CRISPR the bacteria snip out parts of the virus DNA and keep a bit of it behind to help them recognise and defend against the virus next time it attacks.
  • Scientists adapted this system so that it could be used in other cells from animals, including mice and humans.

What other techniques are there for altering genes?

  • Over the years scientists have learned about genetics? and gene function by studying the effects of changes in DNA.
  • If you can create a change in a gene, either in a cell line or a whole organism, it is possible to then study the effect of that change to understand what the function of that gene is.
  • For a long time geneticists used chemicals or radiation to cause mutations. However, they had no way of controlling where in the genome the mutation would occur.
  • For several years scientists have been using ‘gene targeting’ to introduce changes in specific places in the genome, by removing or adding either whole genes or single bases.
  • Traditional gene targeting has been very valuable for studying genes and genetics, however it takes a long time to create a mutation and is fairly expensive.
  • Several ‘gene editing’ technologies have recently been developed to improve gene targeting methods, including CRISPR-Cas systems, transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs).
  • The CRISPR-Cas9 system currently stands out as the fastest, cheapest and most reliable system for ‘editing’ genes.

What are the applications and implications?

  • CRISPR-Cas9 has a lot of potential as a tool for treating a range of medical conditions that have a genetic component, including cancer?, hepatitis B or even high cholesterol.
  • Many of the proposed applications involve editing the genomes of somatic? (non-reproductive) cells but there has been a lot of interest in and debate about the potential to edit germline?(reproductive) cells.
  • Because any changes made in germline cells will be passed on from generation to generation it has important ethical implications.
  • Carrying out gene editing in germline cells is currently illegal in the UK and most other countries.
  • By contrast, the use of CRISPR-Cas9 and other gene editing technologies in somatic cells is uncontroversial. Indeed they have already been used to treat human disease on a small number of exceptional and/or life-threatening cases.

A sperm and egg cell. Carrying out gene editing in germline cells is currently illegal in the UK.

What’s the future of CRISPR-Cas9?

  • It is likely to be many years before CRISPR-Cas9 is used routinely in humans.
  • Much research is still focusing on its use in animal models or isolated human cells, with the aim to eventually use the technology to routinely treat diseases in humans.
  • There is a lot of work focusing on eliminating ‘off-target’ effects, where the CRISPR-Cas9 system cuts at a different gene to the one that was intended to be edited.

Better targeting of CRISPR-Cas9

  • In most cases the guide RNA consists of a specific sequence of 20 bases. These are complementary to the target sequence in the gene to be edited. However, not all 20 bases need to match for the guide RNA to be able to bind.
  • The problem with this is that a sequence with, for example, 19 of the 20 complementary bases may exist somewhere completely different in the genome. This means there is potential for the guide RNA to bind there instead of or as well as at the target sequence.
  • The Cas9 enzyme will then cut at the wrong site and end up introducing a mutation in the wrong location. While this mutation may not matter at all to the individual, it could affect a crucial gene or another important part of the genome.
  • Scientists are keen to find a way to ensure that the CRISPR-Cas9 binds and cuts accurately. Two ways this may be achieved are through:
    • the design of better, more specific guide RNAs using our knowledge of the DNA sequence of the genome and the ‘off-target’ behaviour of different versions of the Cas9-gRNA complex.
    • the use of a Cas9 enzyme that will only cut a single strand of the target DNA rather than the double strand. This means that two Cas9 enzymes and two guide RNAs have to be in the same place for the cut to be made. This reduces the probability of the cut being made in the wrong place.

Scientists Are Creating a Genetic Chainsaw to Hack Superbug DNA to Bits

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E.Coli.

When folks talk about the gene-editing tool CRISPR, they’re usually talking about CRISPR-Cas9, a combination of DNA and enzymes that together act like scissors to cut and paste genes. CRISPR-Cas9 has already been hailed a potential game changer in the fight against cancer, crop pathogens, and environmental problems. But some researchers think a lesser-known flavor of the technology might be the answer to the world’s growing superbug problem. Ladies and gentlemen, meet CRISPR-Cas3.

 Cas9 is in vogue for good reason: It’s a small enzyme that is really good at precisely targeting specific sequences of DNA, making engineering a genome much easier than ever before. Cas3 is terrible at those things. It shreds up the DNA it targets to a point far beyond repair, causing the cell to die. If CRISPR-Cas9 is a genetic scalpel, Cas3 is a chainsaw. Which is exactly why researchers think it might be just the thing to attack the sort of super-tough bacteria that can resist antibiotics.

“What we’re trying to do is kill bacteria,” Rodolphe Barrangou, a molecular biologist at North Carolina State University, told Gizmodo. “It’s like a Pac-Man that’s going to chew up DNA rather than make a clean cut. It chews it up beyond repair. It’s lethal.”

  Barrangou first encountered CRISPR while working for Danisco sequencing Streptococcus thermophilus, a bacteria commonly used in yogurt and cheese production. His early CRISPR work helped lead to the discovery of CRISPR gene editing. Like most scientists in the field, much of his work focused on Cas9. But the clunky, cumbersome Cas3 is a CRISPR enzyme much more common in nature. Barrangou began to wonder whether its boorish nature might be an asset in applications beyond genetic engineering.

In 2015, he co-founded Locus Biosciences, a university spin-off company devoted to reprogramming CRISPR-Cas3 to develop antimicrobials to tackle infectious diseases increasingly resistant to antibiotics, such as C.difficile, E.coliand MRSA. Recently, the company made its public debut after years in stealth mode.

Like Cas9, the Cas3 enzyme can be programmed to target specific DNA, meaning scientists could train it on an unwanted invader. But Cas9 precisely cuts DNA, leaving a double-stranded break that allows the cell to repair itself once the desired edits have been made. Cas3, Barrangou said, is like Pac-Man, chewing up the cell in such a way that leaves it no hope of repair.

“It’s a very promising idea, this had a lot of potential,” Erik Sontheimer, a molecular biologist at University of Massachusetts, told Gizmodo. “Though, I want to caution that when it comes to superbugs, there is no magic bullet.”

Hard-to-kill bacteria, often dubbed “superbugs,” have become a major problem, developing resistance to antibiotics more quickly than we can discover new ones. In a rush to find a solution besides just simply more antibiotics, researchers are experimenting with alternatives, like using predatory bacteria to attack deadly human pathogens.

 Another company, Eligo Biosciences in France, is also focused on using CRISPR to produce antimicrobials. The hope is that not only would it succeed in killing the desired superbugs, but stave off the creation of future superbugs by only targeting one type of bacteria in the body, rather than indiscriminately wiping out many helpful bacteria along the way.

“Antibiotics are indiscriminate—they target all bacteria in the body,” Barrangou said. “If we can use CRISPR to selectively target a particular bacterial genotype and eradicate it, we can leave the rest of the microbiome in tact. It’s like a smart antibiotic.”

Barrangou’s work is in its early stages, but it may be among the most promising alternatives to new antibiotics.

 The company has not yet begun clinical trials, but has had success using CRISPR-Cas3 to fight mice infected with two different strains of E.coli, work Barrangou told Gizmodo it plans to publish later this year. Many challenges remain, including figuring out the best way to actually get CRISPR-Cas3 into the bacteria, with their thick cell walls. There’s also the possibility that pathogens could evolve immunity to CRISPR.

“One of the reasons there is such a crying need for new therapies is that bacteria are very good at evolving ways around whatever we throw at them,” Sontheimer said.

The company will also have to gain FDA approval for any therapy it develops. It hopes that process will be less fraught than it has been for CRISPR-Cas9, since Cas3’s destructive properties mean you can’t make designer babies, superhumans or any other genetically engineered sci-fi catastrophe.

 “We don’t edit a cell and we don’t add anything,” said Barrangou. “But we could kill some bad bacteria.”