MOET-assisted CRISPR/Cas9-mediated MSTN genome editing in caprine embryos by electroporation and embryo transfer

In the world of livestock farming, the goal has long been to improve the animals that provide food and livelihoods for millions. For decades, farmers and scientists have relied on selective breeding, waiting for nature to produce animals with better traits, or using reproductive technologies to multiply the offspring of the best performers. However, this process is slow and limited by the genetic variation already present in the herd. A newer tool, known as genome editing, offers a way to make precise changes to an animal's genetic code, potentially introducing desirable traits much faster than traditional methods could. One specific gene, which acts as a natural brake on muscle growth, has become a primary target for this technology. When this brake is loosened or removed, animals develop more muscle, a trait that is highly valuable for meat production. The challenge lies in delivering the editing tools into the earliest stages of life without harming the delicate embryo, a task that requires a balance of precision and gentleness.

A team of researchers at the Central Institute for Research on Goats in India has taken a significant step toward solving this challenge by combining two established techniques: a method to collect many embryos at once and a way to deliver genetic tools into them without invasive surgery. The researchers focused on the myostatin gene, the natural regulator that limits muscle size in goats. Their aim was to see if they could temporarily disable this gene in goat embryos right after fertilization, using a molecular tool that acts like a pair of scissors to cut the DNA at a specific spot. To do this, they used a technique called electroporation, which uses brief, controlled electrical pulses to open tiny, temporary holes in the cell membrane, allowing the editing tools to slip inside. This approach avoids the need to inject each embryo individually with a needle, a process that is difficult and time-consuming when dealing with many embryos at once.

The study began with eight female goats that were treated with hormones to produce multiple eggs at the same time, a process known as superovulation. The researchers collected seventy-eight fertilized eggs, or presumptive zygotes, from these animals using a minimally invasive surgical procedure. These tiny embryos were then placed in a solution containing the genetic editing tools, which consisted of a protein and a guide molecule designed to find the myostatin gene. The group was subjected to three short electrical pulses, each lasting three milliseconds, to help the tools enter the cells. After the electrical treatment, the embryos were returned to a warm, controlled environment to grow. Remarkably, all seventy-eight embryos developed to the morula stage in culture, with two of them progressing further to become blastocysts, a more advanced stage of development.

To verify that the editing had actually worked, the scientists examined four of the morula-stage embryos. They extracted the genetic material from these cells and looked for signs that the myostatin gene had been cut and repaired with errors, which is how the gene is disabled. The analysis showed that three out of the four embryos, or 75 percent, carried the intended genetic change. This confirmed that the electrical method successfully delivered the editing tools and that the tools functioned correctly inside the developing cells. The researchers then moved to the next critical phase: seeing if these edited embryos could grow into a fetus inside a mother goat. They selected four embryos that had reached the four-cell stage and transferred them into a single female goat that had been synchronized to be ready for pregnancy.

The transfer was successful. The recipient goat became pregnant, and ultrasound scans confirmed the presence of healthy fetuses with beating hearts. Unfortunately, the mother goat died from pneumonia at fifty-seven days of gestation, well before the pregnancy could reach full term. However, the researchers were able to recover two intact fetuses from the uterus. Both fetuses appeared normal in shape and size, with no visible signs of developmental problems, suggesting that the editing process did not harm their growth. The team then tested the genetic material from the ears of these two fetuses to see if the myostatin gene had been edited. The results showed that one of the fetuses, a male, carried the genetic modification, while the female did not. This outcome highlights that even when embryos receive the same treatment, the editing can happen in some but not others, a known variation in this type of science.

This work demonstrates that it is possible to combine the collection of multiple embryos from a donor with a non-invasive electrical delivery method to edit the genome of goats. The study proves that the embryos can survive the electrical pulses, continue to develop in the lab, and successfully implant in a recipient to grow into fetuses. While the editing was not successful in every single case, the fact that the modification was detected in both the early lab-grown embryos and in a fetus that developed inside a mother shows that the approach is viable. The researchers have established a workflow that links the recovery of embryos from a valuable donor, the application of genome editing, and the transfer of those embryos to a surrogate mother. This integrated platform provides a foundation for future efforts to create goats with enhanced muscle growth, offering a more efficient path to improving livestock traits than traditional breeding alone.