Argentine researchers have successfully edited the genes in an equine embryo, raising the possibility of producing horses with custom-designed DNA. “To our knowledge,” they wrote in the paper announcing their work, “edited horse embryos had not been reported until now.”

Working at Kheiron Biotech S.A and FLENI, a non-profit neuroscience research organization, the researchers utilized a technique called clustered regularly interspaced short palindromic repeats (CRISPR) to make genetic alterations in cells that were then used to generate edited cloned embryos.

CRISPR technology is based on the natural ability of bacteria to use ribonucleic acid (RNA) to recognize and delete specific DNA sequences in viruses that attack them. This targeted deletion destroys the virus, protecting the bacteria from infection.

Two scientists—Emmanuelle Charpentier, PhD and Jennifer A. Doudna, PhD— proved in 2012 that these “genetic scissors” can be harnessed and reprogrammed to edit DNA molecules in any organism at any predetermined site. The two received the 2020 Nobel Prize in Chemistry for this discovery, and CRISPR has been used across scientific disciplines to produce a variety of breakthroughs, including the development of mold-resistant crops and cancer-fighting drugs.

Click here to read about genetic tests for horses. 

The researchers in Argentina used CRISPR to “knock out” the myostatin gene. “We chose the myostatin gene because of its role regulating muscle development,” says Gabriel Vichera, PhD, from Kheiron Biotech. “In sport horses this gene plays a crucial role as it controls muscle composition and growth.” More specifically, when this gene is “downregulated”—which can happen as a natural mutation in some individuals—that horse will have more muscle mass, different proportions of muscle fibers and be able to run short distances faster.

“We directed the CRISPR system to the myostatin gene to generate an edition in the DNA sequence that consisted of the loss or gain of some nucleotides that changed the molecule in a way that it cannot be translated to the myostatin protein correctly anymore,” says Lucia Moro, PhD. This effectively Knocked out the gene.

The change was initiated in three lines of fetal fibroblast cells, which were then used as nuclear donors to generate embryos by cloning. These embryos were in vitro cultured until the blastocyst stage which were analyzed for the edition, confirming they were knock-out for the myostatin gene. The embryos were not implanted in mares to develop into foals, but Vichera says they would be expected to have more muscle mass, as is seen in certain breeds of cattle and dogs as well as individual horses where this mutation happens naturally.

Moro says the risk of knocking out the “wrong” gene with CRISPR is very low. “There are many computational programs to design specific gRNA sequences directed to the gene of interest and these programs can determine if there is a risk that the chosen gRNA could alter other gene. In our work, we sequenced two high ranked off-targets in order to know if they were mutated and they were not mutated in our cells.”

The potential applications of CRISPR in horses goes beyond the myostatin gene, says Vichera. “We chose to knock out the myostatin gene as a proof of concept. But our long-term goal is to identify natural advantageous allele sequences present in the genome of some individuals and incorporate them in others to endow them with the desired characteristics.”

CRISPR can also be used to remove or edit disease-causing genes, says Moro. “With this technique available other revisions could be achieved, including the correction of genetic defects that cause equine diseases.”

Reference: “Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer,” Scientific Reports – Nature, September 2020

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