Three years ago, a genome edit (POLLED) was made in two male dairy bulls using transcription activator-like effector nucleases (TALENs) to introduce a hornless phenotype. A new study has investigated whether the POLLED genome edit was faithfully passed to offspring. By crossing one genome-edited dairy bull, homozygous for the dominant POLLED allele, with horned cows, a group at the University of California (UC), Davis, obtained six heterozygous polled calves. The calves had no horns and were otherwise healthy and phenotypically unremarkable. These calves are the first reported offspring of a genome-edited bull.

The researchers have published their findings in the paper “Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull” in the journal Nature Biotechnology. They report that none of the bull’s offspring developed horns, as expected, and blood work and physical exams of the calves found they were all healthy. The researchers also “conducted whole-genome sequencing of all animals using an Illumina HiSeq4000 to achieve ~20× coverage” and analyzed these genomic sequences, looking for any unexpected changes.

In the U.S. dairy cattle industry, the process of disbudding—the destruction of the cells that produce horns before they grow and attach to the skull—is a routine practice. For several reasons, the removal of horns in cattle is useful. For example, hornless cattle cause less injury to other animals, require less feeding trough space, are easier to handle and transport, and have fewer aggressive behaviors. But, the removal of horns is an unpleasant process for the cattle, and many groups have sought alternative, more humane solutions including genetic engineering.

More DNA than expected

All of the group’s data were shared with the U.S. Food and Drug Administration. The FDA’s independent analysis revealed that a fragment of bacterial DNA, used to deliver the hornless trait to the bull, had integrated alongside one of the two hornless alleles in the bull. UC Davis researchers further validated this finding.

“Our study found that two calves inherited the naturally-occurring hornless allele and four calves additionally inherited a fragment of bacterial DNA, known as a plasmid,” said corresponding author Alison Van Eenennaam, PhD, cooperative extension specialist at the UC Davis department of animal science.

Plasmid integration can be addressed by screening and selection, in this case, selecting the two offspring of the genome-edited hornless bull that inherited only the naturally occurring allele.

“This type of screening is routinely done in plant breeding where genome editing frequently involves a step that includes a plasmid integration,” said Van Eenennaam.

Van Eenennaam said the plasmid does not harm the animals, but the integration technically made the genome-edited bull a GMO, because it contained foreign DNA from another species, in this case, a bacterial plasmid.

“We’ve demonstrated that healthy hornless calves with only the intended edit can be produced, and we provided data to help inform the process for evaluating genome-edited animals,” said Van Eenennaam. “Our data indicates the need to screen for plasmid integration when they’re used in the editing process.”

Since the original work in 2013, initiated by the Minnesota-based company Recombinetics, new methods have been developed that no longer use donor template plasmid or other extraneous DNA sequences to bring about introgression of the hornless allele.

Scientists did not observe any other unintended genomic alterations in the calves, and all animals remained healthy during the study period. Neither the bull, nor the calves, entered the food supply as per FDA guidance for genome-edited livestock.

The authors note that “these data can be used to inform conversations in the scientific community, with regulatory authorities and with the public around ‘intentional genomic alterations’ and future regulatory actions regarding genome-edited animals.”

 

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