Are We In Need Of Animal Geneticist

I am in perhaps a somewhat rare position regarding the contemplated USDA regulatory framework, as I actually take several animals from acquiescent species (sheep, cattle) on the footing that were developed using "techniques that use recombinant, synthesized, or amplified nucleic acids to change or create a genome" for "agricultural purposes." I have not published these data and they are the dissertation research projects of graduate students, and so I will only say that these animals were produced using CRISPR/Cas9 genome editing and a sgRNA to produce a gene knockout. This was done by introducing a Cas9-sgRNA ribonucleoprotein (RNP) into unmarried cell zygotes post-obit in-vitro fertilization of in-vitro matured oocytes. The resulting double strand break (DSB) was repaired by the non-homologous end joining (NHEJ) pathway, and subsequent embryo transfer to surrogate dams.

In other words, each animal is a unique individual, and there was no foreign template or intergeneric combination of genetic material introduced into the editing process at whatever fourth dimension. None of the genome-editing target genes were intended to impact animal health, nor was the awarding intended to have an creature health claim. Then as I read through the contemplated regulatory framework, I can't help but personalize how it would affect my research, and how it compares to the existing regulations.

By mode of background, information technology is possibly of import to emphasize that the animals that brand up the inquiry herds and flocks at land-grant universities typically enter the food supply at the cease of experiments. Our dairy cattle eating experimental rations proceed getting milked, the calves from a beefiness cattle crossbreeding study are finished in our feedlot and enter commerce at the end of the study, and our poultry produce eggs that are sold to the public, along with meat products from all our animal facilities, except the horses! We sell foals at the UC Davis foal auction. The food animals are actually candy at our USDA-inspected slaughter facility, and butchered by meat science students. The income from the "meat lab" sales goes dorsum into the operational account for the animal facilities.

And then back to the genome-edited knockout animals I have on campus. Under the existing FDA regulations, they are considered unapproved brute drugs and cannot enter the food supply without a new brute drug blessing or prior authorisation. According to the FDA, I would demand to apply for an investigational new animal drug (INAD) for these animals, then request a Nutrient Use Authority (FUA) for these experimental animals to enter the food supply at the terminate of the experiment. I have really already started to apply for that and the FDA have requested an answer to the following questions earlier they can even open an INAD, let solitary tell me what information I demand to provide to them to potentially obtain an FUA for my experimental factor knockout sheep and cattle:

Clarification of the projection, including the goal of the intentional genomic alterations (IGAs), any relevant information from other animals in which the aforementioned genes have been knocked out (e.grand., references to published articles characterizing knockout mice), and a description of the planned studies
Clarification of how the IGAs were produced, including detailed experimental design
- Include information such every bit which Cas9 nuclease variant was used (e.m., SpCas9, SpCas9-HF1) and any associated information regarding the variant's fidelity, criteria used to design and select guide RNAs, and whatever controls utilized to mitigate the potential for unintended alterations
Available molecular characterization data, including a description of the methods used (due east.thousand., PCR, Sanger sequencing, long-range PCR, etc.)

How does the USDA stack upwardly?

Under the USDA contemplated regulatory framework, published in the Federal Register 12/28/2020, these knockout animals would clearly fit under the "amenable species modified or developed using genetic engineering science and intended for agricultural purposes and human food." If they were knockout plants they would have an up-front exemption from regulation under the SECURE revision as they "issue from natural cellular repair of a targeted DNA pause without any introduced Deoxyribonucleic acid to direct the repair."

For the USDA animal health safety review, I would need to show that "the creature under review is plant to pose no greater risk to animal health than the beast from which it was derived." This is where information technology gets a footling unclear. The term "the brute from which information technology was derived" suggests that the animals are existence derived from a known, unmodified animal, presumably through genetic technology of a cell line derived from that creature, followed by somatic cell nuclear transfer cloning of the edited cells. Just my animals are derived from editing multiple zygotes, so each edited animal has its own unique genomic sequence; there is no animal from which these animals were derived, and conceptually editing outcomes differ between all of these unlike individuals, especially considering the double strand break was repaired using the error-prone NHEJ pathway. So does each individual animal accept to have a dissimilar evaluation?

The rubber review requires "a molecular characterization of the modification and an agreement of the procedure by which it was introduced, that the intended alter was made and that there were no unintended disruptions of endogenous genes, unintended DNA insertions, or off-target changes if the genome was modified without inserting Deoxyribonucleic acid."

It is not hard to prove that the intended alter was made, we typically practice that with a PCR amplification of the target locus, and sequencing of the amplified PCR product. But to answer the rest of those questions will require, at a minimum, whole genome sequencing of every animal, and proving a negative, proving no unintended disruptions of endogenous genes, unintended Deoxyribonucleic acid insertions, or off-target changes if the genome was modified without inserting DNA. Both the sequencing and the bioinformatics to attempt to respond these questions would be very expensive, well beyond the cost of a sheep, or even a moo-cow. This is particularly true when every beast has a unique genotype, and there is no way to differentiate off-target changes from spontaneous de novo mutations.

suckler cow and calfs x — Credit: Agriland

Further, at a minimum, the beast health risk assessment would include an evaluation of the post-obit issues:

Molecular Label: What is the genetic modification(s) in the brute, how was the genetic modification(s) introduced, and how does the genetic modification(s) alter protein or ribonucleic acid (RNA expression)?
Brute Health: Is there scientific evidence that the modified animal could plausibly, either directly or indirectly, increase susceptibility of livestock, including of the animal itself, to pests, non-infectious diseases, or infectious diseases of livestock, including zoonotic diseases? Is in that location scientific bear witness that the modified animate being could plausibly increase the spread of pests or infectious diseases of livestock, including zoonotic diseases? When a plausible pathway to such an increased gamble is identified, further analysis would be conducted to evaluate the pathway. When an animal wellness claim is fabricated or a modification is known to adversely impact creature health, the review would assess the animal wellness claim.
Environmental Factors: Is there scientific evidence that introduction of the modified animal into the environment may result in environmental impacts that would warrant review pursuant to the National Ecology Policy Act (NEPA) or other statutes?

And in addition, the FSIS review would include an evaluation of the post-obit problems (theoretically pre-slaughter, although obtaining meat for compositional analysis from animals pre-slaughter is manifestly problematic):

Evaluation of expressed substances: Is there scientific evidence that the genetic modification could upshot, directly or indirectly, in toxins, chemical residues, or other potentially deleterious substances in meat or poultry products?
Allergenicity: Is there scientific evidence that the genetic modification would directly or indirectly alter the allergenic potential of meat or poultry products derived from the brute?
Food storage and processing: Is there scientific evidence that meat or poultry products derived from the modified beast could mislead consumers regarding wholesomeness or the need for advisable storage (e.thousand., meat that maintains a red advent even when spoiled)?
Compositional analyses of key components: Is there scientific bear witness that meat or poultry products from the modified animal are compositionally (e.thousand., nutritionally or functionally) no different than meat from conventional animals, such that it meets any regulatory definition, standard of identity or other labeling requirement, and consumer expectations for the applicable product?

Needless to say, providing answers to all of these questions for each private knockout animal would be an insurmountable hurdle for an bookish laboratory. Throughout the USDA contemplated regulatory framework the term "developer" is used. For case, "Under the contemplated regulatory framework, developers could request that USDA conduct a take chances-based and science-based prophylactic review focused on animal health." Historically, developers of genetically engineered plants have been big multinational companies who have the resource to provide the regulatory information to support commercialization and ultimately deregulation of their products. Inquiry universities are not developers in this sense, but nosotros yet produce acquiescent species modified using genetic applied science and intended for agronomical purposes and man food.

One of the questions the USDA poses in the request for comments is,

"How frequently does a start-up company or not-for-profit university or inquiry organisation modify or develop an animal using genetic technology?"

My answer would be, "never." Researchers at universities often modify animals using genetic engineering, just never has one adult and commercialized an "amenable species modified or adult using genetic engineering and intended for agricultural purposes and man food." The two food approvals for genetically engineered animals were both obtained by companies, admitting small companies. AquaBounty for the "AquAdvantage" salmon, and Revivicor for the "GalSafe" pig. To appointment, neither of these products has been sold for food in the United states of america, although AquaBounty is getting mighty close.

Uncertain future

In closing, as long as animals produced using genetic applied science, fifty-fifty those that could have been produced using conventional breeding, are subjected to unique regulatory scrutiny not required of identical products produced using conventional convenance, research in food animals using genetic engineering science for agronomical applications will be cost-prohibitive in the United states of america.

Another question the USDA poses is,

"Should USDA exempt certain types of genetic modifications of acquiescent species intended for agricultural utilise from regulation? If so, what types of modifications and why?"

I would suggest that the SECURE up-forepart exemptions for certain types of modifications in plants, specifically, a modification (ane) resulting from natural cellular repair of a targeted Deoxyribonucleic acid break without any introduced Deoxyribonucleic acid to direct the repair; (2) a targeted single base pair substitution; or (three) a modification that introduces a gene known to occur in the plant'due south factor pool, or causes a change in a factor that corresponds to a version of that gene present in the organism'south gene pool should exist extended to genetically engineered animals. These exemptions were primarily designed to exclude from regulatory oversight organisms that could have been achieved using conventional convenance. Just like the Coordinated Framework calls for.

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If y'all accept comments on the contemplated regulatory framework, there is a 60 day public annotate period that closes two/26/2021. I would encourage all people working in this field to post comments. Comments can exist posted at the Federal eRulemaking Portal .Supporting documents and any comments that are received on this Advance Notice of Proposed Rulemaking may be viewed here.

In the words of one commentator:

"It, of form, remains to be seen what volition be the Biden Administration's arroyo to ag biotech; however, given the consistency of approach to the evolving ag biotech regulatory structure past both the Obama and Trump Administrations, it would not be surprising if the Biden Administration too continued the evolution of a more science-based, bear witness-based, and risk-based arroyo to regulation of ag biotech – including genetically engineered animals. Information technology would behoove stakeholders that would support such consistency of regulatory approach to submit detailed comments that would assistance USDA in developing such a notice of proposed rulemaking (NPRM).

As with all opportunities to provide comments on Federal agency proposals, it is important to submit concise, substantive, and well-supported and well-documented comments to the administrative docket. In this case, given the trans-Administrations nature of the activity, it is important that interested stakeholders utilize this opportunity to best advantage to urge the Biden Administration to go along a 21st Century arroyo to regulating ag biotech."

– Keith Matthews, Wiley Rein LLP

Alison Van Eenennaam is an extension specialist in animal biotechnology and genomics, Department of Brute Science, Academy of California, Davis.

A version of this article was originally posted at the BioBeef Blog and has been reposted here with permission. BioBeef can be institute on Twitter @BioBeef

Source: https://geneticliteracyproject.org/2021/01/18/can-biden-usda-bring-americas-animal-gene-editing-rules-into-the-21st-century/

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