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By Claire Robinson and Dr. Michael Antoniou

A concept patent has been filed for a method that includes the use of CRISPR/Cas-mediated gene editing to generate transgenic birds so that no male offspring are able to hatch.

Israeli scientists led by Dr. Yuval Cinnamon (named as an inventor on the patent) are proposing this method to be used in chickens so that only female chicks will hatch, which will then go on to become laying hens for egg production.

Currently, male chicks are killed when young as they are of no use to the egg or chicken meat industries. Even then, hatching the male chicks and keeping them alive until they are killed is viewed by the industry as a waste of energy and other resources.

More seriously, the practice of killing male chicks is an animal welfare issue.

In this light, the transgenic CRISPR-edited chickens are being hyped as a boon for animal welfare, on the grounds (in the BBC’s words) that the technology could “prevent the slaughter of millions of male chickens in the U.K., which are culled because they don’t lay eggs.”

But our investigation shows that such claims are disingenuous in the extreme. In fact, the technology forces mother hens to pass on a lethality (killer) gene, which is intended to kill all male embryos before they hatch from the egg.

The genes that are most reliably lethal, and therefore most likely to be used, produce highly toxic proteins. The hen should only produce the toxic protein under the influence of blue light, according to the patent.

However, if the technology doesn’t work perfectly, the founder breeder hens and their egg-laying daughters could produce a toxin at low levels in their bodies, leading to health problems in these chickens.

The male chick embryos killed successfully with the lethality gene could, depending on the particular gene used, effectively be toxic waste and could not be put into the animal feed supply — the current destination for unwanted male chickens.

And the lethality gene could escape into the environment or into bacteria, and again, depending on the gene used, could endanger humans, animals and wildlife.

Moreover, there appears to be no proof that the technology will work as intended, as there is no evidence in the public domain that a live transgenic breeder hen has actually been produced. The experiments described in the patent are all done on cells in test tubes/flasks (in vitro) or on the egg (in ovo).

In spite of all this, the European Commission has rushed to assure the developers that the egg-laying hens and their eggs are not genetically modified organisms (GMOs) and can therefore be sold without safety checks and GMO labeling.

The method

CRISPR/Cas gene editing is used in an SDN-3 (gene insertion) procedure to target the integration of a transgene (a foreign gene, in this case, the lethality gene) into the male sex Z chromosome, with the egg-laying hen passing on that transgene to all male embryos of the next generation of chickens.

On exposing the eggs to blue light, the lethality gene is activated and kills the male embryos before they hatch.

Lethality gene is likely to produce highly toxic protein

In order to ensure the reliable killing of the male chick embryos at an early stage of their development, the lethality gene that the developers insert will have to be highly toxic.

The various lethality-inducing proteins mentioned in the patent that are supposed to work by inhibiting growth/development (paragraphs 0156, 0157) or essential signaling pathways, such as “bone morphogenetic protein antagonist” or “RNA-guided DNA endonuclease enzyme” (paragraphs 0159, 0160), may be too uncertain in their effects.

Therefore the developer will almost certainly choose to use a known highly toxic element — such as genes encoding for diphtheria toxin or ricin toxin, both of which are specifically mentioned in paragraph 0158 as possible candidates for the lethal gene.

The fact that the authors illustrate their concept using a diphtheria toxin lethality gene, albeit within the context of in vitro tissue culture cell experiments (Figure 24A), supports this line of thinking.

A gene encoding cholera toxin, another highly toxic poison, could conceivably be used, as the patent does not restrict the lethal gene to certain named types.

This raises the question of how “tight” and foolproof the expression of the lethality gene cassette is — in other words, whether it is completely silent as desired until activation by blue light illumination, or whether there is some low but significant expression prior to blue light illumination.

Indeed, evidence of lethality gene expression leakiness is provided in Figure 13 of the patent (upper panels). It is common knowledge that all transgenic systems are leaky — it’s only a question of degree.

Thus the optogenetic (blue light) activation system linked to the lethality gene cassette will almost certainly be “leaky.” This means that in the female founder breeding hens, even in the absence of blue light, the lethal gene may not be silent.

So these female founder breeding hens and their egg-laying female offspring could express the lethality gene at a low level. This would mean that these hens would be producing a lethal toxin inside their bodies. As a result, they could suffer health problems.

This possibility (which is far from unlikely) raises welfare questions about the health of the female founder hens and their female offspring. Their health status will depend on the nature of the lethality gene and to what extent it expresses in their bodies.

This is a major ethical issue, beginning with the action of genetically engineering a mother hen to pass a killer gene to all her male offspring.

As mentioned above, the lethal toxin-generating gene could escape into the environment or into bacteria. If it gets into bacteria, it could transfer from the bacteria into people or animals, with potentially serious consequences to their health.

Any male embryos that are killed using a toxic lethality gene will need to be treated as toxic waste and could not be used, for example, as animal feed, which is the usual destination for rejected male embryos or chicks in the non-GMO egg industry.

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Proof-of-concept only

It is important to note that the experimental data presented in the patent application only attempt to demonstrate the feasibility of the various components of the method. They have tested all these components separately, but do not actually demonstrate that they can be brought together to produce a female transgenic founder chicken that can be used for breeding egg-laying hens.

A search of scientific databases also failed to identify a transgenic breeding hen of the type that the method aims to generate. Thus based on current publicly available information, a transgenic live breeder chicken of the type described in the patent does not exist.

The patent is a method patent that tries to provide proof-of-concept and only describes in vitro and in ovo experiments. At most, these experiments show that exposure to blue light can activate gene expression as desired in vitro and in ovo.

They also show the killing of tissue culture cells using the lethality gene system. They show protein synthesis inhibition from the expression of the diphtheria gene (but not strictly cell or embryo death) in ovo — but not through activation by blue light. They show the killing of tissue culture cells with diphtheria or caspase (cell death-inducing) genes, but again, not via blue light activation.

At best they show that in ovo injection of a growth inhibitor protein (noggin) can arrest embryo development at an early (blastomere) stage. No doubt the idea is that if you can express these toxic proteins from a gene via blue light illumination, then it could work.

But there’s no proof that it does.

EU Commission claims the laying hens and their eggs are not GMOs

The EU Commission wrote to the German Federal Office of Consumer Protection and Food Safety (BVL) in July 2021, stating that the laying hens resulting from this genetic modification process and their eggs are not GMOs and would not fall under the EU’s GMO regulations.

The EU Commission reaches its conclusion based on the supposed absence of the transgene (or fragments thereof) in the female hens. However, and crucially, the EU Commission is grossly misinterpreting the law.

The EU definition of a GMO is not an organism that contains transgenes, but an organism “in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination.”

The law does not state that transgenes have to be present in order for an organism to be classed as a GMO.

There is no proof that the female hens in question are free from such unnatural genetic alterations, as described in this legal text. Therefore the Commission appears to be acting against the law.

Do the female laying hens contain transgenes?

Let’s suppose for a moment that the EU law was worded completely differently from how it is and that it does define a GMO as an organism containing transgenes. Even in this imaginary scenario, it is not valid for the Commission to assume that the female hens do not contain unintended transgenes in part or in whole.

Scientific evidence in plants and human cells shows that fragments of foreign DNA from the gene-editing tool can inadvertently integrate into the genome during the gene-editing process and end up scattered across the genome.

In the case of the transgenic chickens, in order to produce the founder hens, integration of the lethality gene is targeted into the male sex-determining Z chromosome, using the CRISPR/Cas gene-editing tool.

But this process may not go as planned.

While the lethality gene cassette may end up in the intended location on the Z chromosome, fragments of the lethality gene or the plasmid DNA molecules encoding the CRISPR/Cas tool may also integrate in other regions of the genome – that is, on chromosomes that will be passed down to both male and female chicks.

As a result, both the founder hens and their female egg-laying offspring could inadvertently contain fragments of the CRISPR gene-editing tool and/or fragments of transgenes in their genomes.

There appears to be no published evidence showing that this procedure does not give rise to inadvertent transgene fragment integration and that the resulting transgenic founder hens and their female offspring are free from such foreign DNA. Moreover, existing evidence suggests that this is highly likely to happen.

The way to find out if it has happened is to do a genomics (whole genome sequencing) analysis of the founder hens and their female egg-laying offspring. But this basic investigation may not at present be possible if, as appears, the desired transgenic founder hen does not exist in actuality.

So until evidence is provided to the contrary, we can assume that fragments of the CRISPR gene-editing tool and/or fragments of transgenes may have integrated into their genomes. If this is the case, then the founder hens and their offspring, the female egg-laying hens, will be transgenic, as will be the eggs of the laying hens.

Therefore even under a hypothetical law that defined a GMO as an organism that contains transgenes, all three would have to be labeled as GM. The Commission would therefore be acting against this hypothetical law — and against its own incorrect interpretation of the law — in stating that the laying hens and their eggs are not GMOs.

Under EU law, the egg-laying hens and their eggs are certainly GMOs, though not because of the possible presence of transgenes.

As Testbiotech explains:

“In the case of the laying hens, they are the direct female offspring (F1) of the transgenic chickens.

“They inherit (regardless of whether the transgene works as supposed) genetic material from the mother hens which also will be transferred to the eggs. Thus, there can be no doubt that the laying hens and the eggs produced are products of GMOs and consist of GMOs.

“As can be seen with oil, starch or sugar produced from GM plants, it is the production process which is the decisive criterion for the implementation of EU law and not the presence of genetically modified material [e.g. transgenes] in the end product.”

The Commission’s action in sending the BVL a letter stating that these animals are non-GMO shows not only its misinterpretation of EU law but also that it accepts the GMO industry’s self-declaration of transgene-free status, without requiring any proof.

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Unintended genetic changes

The gene-edited founder breeding hens are likely to have unintended changes in their genome, such as insertions, deletions or rearrangements of DNA, at both the intended edit site (on-target) and at other locations in the genome (off-target).

This could lead to disturbances in patterns of gene function which could lead to health or welfare issues in the chickens. Even if at the site of insertion of the lethality gene, all is as intended without any unwanted mutations, unintended genetic alterations at off-target sites will be passed on to the egg-laying daughters of the founder breeding hens.

It is not known how carefully the developers will look for such unintended effects — only long-read whole genome sequencing and subsequent “omics” molecular analysis of the chickens will suffice — and how carefully they will try to breed them out.

Any unintended effects that are not bred out will be passed down to the egg-laying hens. Without strict regulation requiring such examinations, it is uncertain that they will be undertaken.

What is the Commission actually deregulating?

In sum, there appears to be no available information on how any live GM chickens were generated and indeed if they were generated at all.

So the Commission appears to be acting beyond its expertise, as well as beyond its mandate, in its rush to deregulate something that may not work; may not exist in a utilizable form, and if it does, will likely not be as free from transgenic material as the Commission assumes; and may cause serious public health and environmental problems, as well as severe health or welfare issues for the chickens themselves.

Alternatives are available

While unwanted male chick embryos are commonly killed by gassing them or grinding them up alive, more humane alternatives are available. These alternatives seem to be preferable to a potentially dangerous gene editing route using lethality genes.

One such alternative technology is egg screening using the Sellegt method, which enables producers to sex the chick embryos on day nine of incubation and selects out the unwanted males.

Eggs produced using this method are already being sold by supermarkets under the label “Respeggt,” which promises that the eggs are “free of chick culling.” Other already-available sex determination methods are described on the Wikipedia page on in ovo sexing.

It may be argued that the patent for the gene-edited birds allows male embryos to be killed using exposure to blue light before the nine-day point at which the Sellegt method becomes viable — though this raises the question of whether a nine-day-old embryo is any more sentient than a 1-8 day-old embryo and therefore if there is any moral gain in using the gene-editing system because it theoretically allows for earlier killing.

The problem with this argument is that the patent hedges its bets and also claims that the killing point can be any time between one day and the full 21-day egg incubation period.

So it cannot be assumed that killing a male embryo with a lethality gene is in any way more humane (on the grounds that it takes place at an earlier stage) than existing alternative non-GMO methods — and the latter does not result in a potentially toxic product.

This patent is under consideration by the European Patent Office but has not yet been granted. In deciding whether to grant patents, patent offices must consider three things: novelty, a non-obvious inventive step, and utility.

If the toxic lethality gene is only activated at 10–21 days of incubation, as is provided for in the patent, then the Patent Office would be justified in refusing the application, as the technology described is not an improvement on existing technologies and therefore has no utility.

Dual-use chickens: A more humane and sustainable option?

For those who object to any killing of male chick embryos on the grounds of animal welfare or waste, but wish to see chicken meat production continue, another option is available that would enable the raising to maturity of the males.

That is dual-use chickens, in which the females serve as egg layers and the males as meat. Such chickens are commonly available but are not commonly used in the chicken meat industry because males do not put on weight as quickly as females.

So by separating out breeds between egg-laying and meat-producing, productivity is arguably being prioritized over animal welfare and sustainability.

Conclusion

In conclusion, this gene-editing application appears to be:

  • Of unknown efficacy in producing the intended gene-edited live chickens.
  • Potentially dangerous for the chickens themselves, raising animal welfare concerns.
  • Potentially dangerous for humans and other animals, who may be exposed to escaped lethality genes expressing highly potent toxic protein products (e.g., diphtheria, ricin, or cholera), due to the envisaged large scale use of this technology. These toxin-encoding lethality genes and their toxic protein products could also put at risk the environment as a whole.
  • Ethically questionable. The developers are genetically engineering a mother hen to pass a killer gene to all her male offspring when there are already-available alternatives, such as egg sexing early in the incubation period.
  • Of doubtful utility, since it seems not to provide any more humane or efficient system of preventing the birth of male chicks than is already available via other technologies.

In addition, the egg-laying hens and their eggs are GMOs under EU law.

Therefore the European Commission should correct its advice to the German regulator and state that these GM products should be subjected to a risk assessment and GMO labeling.

Originally published by GMWatch.

Claire Robinson, MPhil, is an editor at GMWatch, a news and public information service on issues relating to genetic modification. She was formerly the research director at the sustainability nonprofit Earth Open Source and is a co-author of the book “GMO Myths and Truths.”

Michael Antoniou, Ph.D., is a reader in molecular genetics and head of the Gene Expression and Therapy Group, Department of Medical and Molecular Genetics, King’s College London School of Medicine, UK. He has 34 years’ experience of using genetic engineering technology, including genome-editing tools, in the investigation of gene organization and control, with more than 100 peer-reviewed publications. He holds inventor status on gene expression biotechnology patents. His discoveries in gene control mechanisms are being used for research as well as the development of diagnostic and therapeutic products for genetic disorders. Antoniou has taken an active interest in the safety of GM crops and their associated pesticides. In recent years he has published papers demonstrating the non-equivalence of glyphosate-tolerant GM corn and non-GM corn and severe kidney and liver toxicity from chronic exposure to an ultra-low dose of Roundup.