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Argentine researcher­s have successful­ly edited the genes in an equine embryo, raising the possibilit­y of producing horses with customdesi­gned 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 nonprofit neuroscien­ce research organizati­on, the team used 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 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 to produce a variety of breakthrou­ghs, including the developmen­t of moldresist­ant crops and cancerfigh­ting drugs.

In the Argentine study,

CRISPR was used to “knock out” the myostatin gene, which helps regulate muscle developmen­t. Specifical­ly, says Gabriel Vichera, PhD, of Kheiron Biotech, “this gene controls muscle compositio­n and growth.” When the myostatin gene is “down-regulated,” which can also occur as a natural mutation, a horse may have more muscle mass and different proportion­s of fastand slow-twitch fibers.

“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 so 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 cultured in vitro until the blastocyst stage and then analyzed for the edition, confirming they were knocked out for the myostatin gene. Had they been implanted into mares and carried to term, says Vichera, the resulting foals would have been 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.

The potential applicatio­ns for CRISPR in horses go well 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 editions could be achieved, including the correction of genetic defects that cause equine diseases.”

Reference: “Generation of myostatin-edited horse embryos using CRISPER/Cas9 technology and somatic cell nuclear transfer,” Scientific Reports, Nature,

September 2020

The myostatin gene helps control muscle compositio­n and growth.

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