THIRD WORLD NETWORK BIOSAFETY INFORMATION SERVICE
Dear Friends and Colleagues
Genome-Edited Hornless Cattle Found to Have Unintended Antibiotic Resistance Genes
Genome-edited hornless cattle were produced in 2016 by Recombinetics, Inc. of Minnesota. The company reported that “our animals are free of off-target events”. But in a paper published online on 28 July 2019, US Food and Drug Administration (FDA) researchers re-examined the DNA of the genetically dehorned calves and found that the two calves’ genomes did contain unintended DNA alterations.
One calf was found by FDA to have an unintended duplication of the polled gene locus; while the DNA of both calves contained two antibiotic resistance genes, along with various other gene sequences of bacterial origin. The inadvertently introduced bacterial sequences were found close to the editing site. Of the two antibiotic resistance genes found by FDA, one confers Neomycin/Kanamycin resistance and the other Ampicillin resistance. The unexpected DNA sequences detected by the FDA researchers originate from the plasmid (a DNA carrier) used by Recombinetics to introduce the polled DNA sequence.
The presence of the previously undetected antibiotic resistance genes in genome-edited cattle raises issues of biosafety given that there is a strong global push to limit the spread of genes conferring antibiotic resistance. No research has been carried out on the possible consequences for animal health, or whether these additional genes are biologically active.
The finding that genome-editing techniques can, unbeknownst to the developer, introduce foreign DNA is a significant blow to the no-regulation argument. As the genome-edited cattle do contain DNA unnatural to cattle, despite the claims of their developers to the contrary, this makes them subject to FDA regulation. This finding is also a powerful vindication of the EU’s stand to regulate genome-edited organisms as GMOs.
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Item 1
FDA FINDS UNEXPECTED ANTIBIOTIC RESISTANCE GENES IN ‘GENE-EDITED’ DEHORNED CATTLE
Jonathan Latham, PhD and Allison Wilson, PhD
Independent Science News
12 August 2019
https://www.independentsciencenews.org/news/fda-finds-unexpected-antibiotic-resistance-genes-in-gene-edited-dehorned-cattle/
Gene-editing is seen by many as the ultimate in precision breeding. Polled cattle, whose horns have been genetically removed, have been presented as exemplars of this–a socially beneficial use of precise genome engineering. Such hornless cattle were produced in 2016 by Recombinetics, Inc., of St. Paul, Minnesota, a development that was reported in the journal Nature Biotechnology (Carlson et al, 2016).
In that publication, Recombinetics researchers reported detecting no unexpected alterations, such as insertions or deletions of DNA, as a result of the gene-editing procedure. They concluded “our animals are free of off-target events” (Carlson et al, 2016).
The Carlson claim, along with other assertions of precision, have comprised the major ingredient of a larger global argument, led by the biotech sector, in favour of light (or no) oversight of animals and plants produced through gene-editing.
Thus, in a commentary titled ‘Regulate genome-edited products, not genome editing itself‘ which accompanied the Carlson publication, academic researchers led by Dana Carroll, some of whom are associated with Recombinetics, stated:
“Any mutations leading to obviously deleterious phenotypes would be eliminated from breeding programs. Other hypothetical risks, such as a modified protein that turned out to be allergenic to humans, might equally well arise naturally in the absence of human intervention. The effects of genome editing are largely identical to those of the natural processes that continually create variation in the genomes of food animals. From this point of view, it is hard to see why the process of genome editing to introduce defined genetic changes should be regulated” (Carroll et al., 2016)
But, in a paper just published online (July 28th, 2019), Food and Drug Administration (FDA) researchers re-examined the DNA of the genetically dehorned calves, whose genome sequences had been posted online by Recombinetics (Norris et al., 2019). In fact, the two calves’ genomes did contain unintended DNA alterations.
One calf was found by FDA to have an unintended duplication of the polled gene locus; while the DNA of both calves contained two antibiotic resistance genes, along with various other gene sequences of bacterial origin. The inadvertently introduced bacterial sequences were found close to the editing site. Of the two antibiotic resistance genes found by FDA, one confers Neomycin/Kanamycin resistance and the other Ampicillin resistance.
[The new FDA research is posted on the bioxriv server as a preprint (https://www.biorxiv.org/content/10.1101/715482v1).]
The presence of the previously undetected antibiotic resistance genes in gene-edited cattle raises issues of biosafety given that there is a strong global push to limit the spread of genes conferring antibiotic resistance. This is because every cell of the gene-edited cattle with the polled locus will also contain the resistance genes, allowing them to easily be transferred to bacteria.
The FDA researchers’ discovery of bacterial DNA in the genome of the calves raises more general regulatory questions about gene-editing too, beyond a lack of precision.
The unexpected DNA sequences detected by the FDA researchers originate from the plasmid (a DNA carrier) used by Recombinetics to introduce the polled DNA sequence. As the commentators themselves, arguing for regulatory exemption, proposed in 2016:
“Given that the United States has no specific legislation regulating animal breeding, there would appear to be no authority for the FDA to regulate varieties that carry naturally occurring alleles produced using genome editing”. (Carroll et al., 2016)
But what the new FDA finding demonstrates is that the Recombinetics gene-edited cattle do contain DNA unnatural to cattle, despite the claims of their developers to the contrary. Thus FDA does have the authority to regulate.
Other researchers have in the past called on genetic engineers to rigorously exclude potential contaminating DNA, such as unwanted bacterial plasmid DNA, as part of standard experimental procedures (Wilson et al., 2006). This, Recombinetics apparently failed to do.
FDA’s demonstration that gene-editing techniques can, unbeknownst to the developer, introduce foreign DNA is likely to be seen as a significant blow to the no-regulation argument. It is also a powerful vindication of the EU approach, which is to regulate gene-edited organisms as GMOs.
References
Daniel F Carlson, Cheryl A Lancto, Bin Zang, Eui-Soo Kim, Mark Walton, David Oldeschulte, Christopher Seabury, Tad S Sonstegard & Scott C Fahrenkrug (2016) Production of hornless dairy cattle from genome-edited cell lines. Nature Biotechnology 34, pages 479–481.
Carroll, D., Van Eenennaam, A. L., Taylor, J. F., Seger, J., & Voytas, D. F. (2016). Regulate genome-edited products, not genome editing itself. Nature Biotechnology 34: 477–479.
Norris, Alexis. L., Stella S. Lee, Kevin J. Greenlees, Daniel A. Tadesse, Mayumi F. Miller, Heather Lombardi (2019) Template plasmid integration in germline genome-edited cattle. doi: https://doi.org/10.1101/715482
Wilson, Allison K., Jonathan R. Latham, and Ricarda A. Steinbrecher (2006) Transformation-induced mutations in transgenic plants: analysis and biosafety implications. Biotechnology and Genetic Engineering Reviews 23.1 : 209-238.
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Item 2
TEMPLATE PLASMID INTEGRATION IN GERMLINE GENOME-EDITED CATTLE
Alexis L. Norris, Stella S. Lee, Kevin J. Greenlees, Daniel A. Tadesse, Mayumi F. Miller, Heather Lombardi
Biorxiv
28 July 2019
doi: https://doi.org/10.1101/715482
https://www.biorxiv.org/content/10.1101/715482v1
Abstract
We analyzed publicly available whole genome sequencing data from cattle which were germline genome-edited to introduce polledness. Our analysis discovered the unintended heterozygous integration of the plasmid and a second copy of the repair template sequence, at the target site. Our finding underscores the importance of employing screening methods suited to reliably detect the unintended integration of plasmids and multiple template copies.
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Item 3
GENETICALLY ENGINEERED HORNLESS CATTLE: FLAWS IN THE GENOME OVERLOOKED
Testbiotech
6 Aug 2019
https://www.testbiotech.org/en/news/genetically-engineered-hornless-cattle-flaws-genome-overlooked
New techniques for genetic engineering not as precise as claimed
According to research undertaken by experts at the US Food and Drug Administration (FDA), gene-editing errors in the genome of the animals are, in many cases, often being overlooked. This was the finding from the FDA genome analyses of cattle genetically engineered not to grow horns. The animals were genetically engineered by Recombinetics; the company also filed a patent on the genetically engineered cattle. The cattle have for some years been held up and presented as a positive example for the application of new genetic engineering techniques. However, it appears to have so far gone unnoticed that the gene-editing has resulted in major unintended effects.
The gene scissors (nucleases) used in this case are known as TALENs; a method frequently described as highly precise. However, as the FDA research shows, apart from the desired gene sequences being inserted into the genome, DNA originating from genetically engineered bacteria used in the process was also inserted. The researchers found, amongst others, complete DNA sequences that confer antibiotic resistance in the genome of the cattle. No research has been carried out on the possible consequences for animal health, or whether these additional genes are biologically active.
As the experts from the FDA point out, the errors caused by the genetic engineering technique are unlikely to be individual cases. So far, the methods used to search for unintended effects in GE animals are frequently inadequate, and are used even though much better technologies are available.
Recombinetics has already applied for more than a dozen patents on genetically engineered animals. The large breeding corporates, Genus and Hendrix Genetics, might be amongst those profiting from the introduction of these animals on the market. Both companies have contracted and funded the work of Recombinetics and its affiliate Acceligen. In addition to polled cattle, Recombinetics is moving to develop a precision gene-editing breeding method to eliminate the need to castrate pigs. Alongside questions about safety and ethics, there are urgent questions that need to be answered regarding the application of gene-editing techniques, which in general lead to massive problems in animal welfare.
Contact:
Christoph Then, info@testbiotech.org; Tel. +49 151 54638040