Gut health: CRISPR-based Kill Switch causes bacteria to self-destruct

Researchers from Washington University in St. Louis have genetically engineered a kill switch that causes bacteria to self-destruct after reaching their goal.

A article describing the research titled “Genetically Stable CRISPR-Based Kill Switches for Engineered Microbes” was recently published by Nature Communications.

“To my knowledge, this is the best circuit breaker ever developed,” said Tae Seok Moon, co-author of the study and an associate professor in the university’s Department of Energy, Environmental and Chemical Engineering, during a meeting. an interview with BioSpace.

Although Moon’s background is primarily in chemistry and bioengineering, he became personally invested in gut health after suffering severe and life-threatening symptoms himself. “At that time, I thought that even though I was dying, I wanted to make a contribution to the world with my research,” he shared. “Now, after my miraculous recovery, I want to continue my research journey with the ultimate goal of helping people.”

The article states that Escherichia coli Nissle 1917 (EcN), the probiotic strain used in the research, is one of the most commonly modified strains. E.coli can be found in the intestinal tract of humans and animals and works to keep the gut healthy.

“Modified EcN strains have been used successfully to diagnose and treat bacterial infections, cancers, gastrointestinal bleeding, inflammatory disorders, and obesity in a variety of animal models,” the article continues. Nevertheless, probiotics are living organisms that can mutate, which introduces specific safety issues in their medical applications, requiring special attention to biocontainment.

Biocontainment has always been an area of ​​great interest to Moon. “Biocontainment is very important. We don’t know the consequences of the bacteria once they are released into the environment. When developing genetically modified microbes, or GEMs, we need to be aware of the consequences. We have to have ways to control them,” he said. “I would like to say that I am cruel to bacteria, as are all humans. I want them to self-destruct once their mission is complete. This is the basic idea of ​​the whole article.

The engineering team used a mouse model to achieve their goal of destroying the bad bacteria in the mouse gut while preserving the good bacteria. “We wanted to develop a so-called biosensor that is housed in bacteria,” Moon said. “Bacteria are very small. They perceive small changes in their environment, such as changes in temperature. If they become the bad guys, we’ll feed the bacteria a chemical, inducing the kill switch to make them self-destruct and prevent the bacteria’s negative impacts on the environment or humans.

In the first stage of the research, engineers used a chemical droplet to eliminate gut bacteria from mice. “During this step, we were able to completely eliminate the bacteria from the mice’s gut when we gave them the chemical. However, if we didn’t give them the chemical, the bacteria still survived. The problem with this kill-on-demand strategy is that the bacteria would exit the body and potentially get out of control,” Moon explained.

To combat this, the team looked at the differences between a body and the outside world.

“The main difference between our body and our environment is temperature. A constant body temperature is very important for humans. If the body temperature increases slightly or decreases slightly, we could die,” Moon said. “Considering this, we developed a temperature sensor in probiotic bacteria. They can sense a slight difference in temperature, which triggers the circuit breaker, and then they self-destruct at environmental temperatures that would typically be 34oC or less. We were able to demonstrate this “cold” temperature-induced kill switch in the mouse model.

The next step was to measure the stability of the circuit breaker. “Mutation is the number one enemy of bioengineers. Mutation happens all the time. Bacteria don’t want to die. Their only purpose is to reproduce and divide. Because of this, the team, including study co-author Austin G. Rottinghaus, had to repeatedly adapt to the changes and improve their kill switch. “Every time we upgraded the kill switch, another mutation would occur that made the switch non-functional,” Moon explained. “My student, Austin, has been amazing through this process. He never gives up.”

The idea of ​​a backup generator became the ultimate solution for the team. “If your power goes out, you rely on a backup generator. With that in mind, we improved the kill switch, ultimately using four kill switches in total to make it functionally stable enough.

Next, the team was challenged to get the bacteria to work properly in the mouse gut. To solve this problem, they introduced a control strain.

“When we gave both temperature and chemical intake, the bacteria containing the kill switch almost always died. The kill switch is potentially applicable to all microbes given to humans, dogs, cows, pigs, etc. ., based on practical applications ranging from the destruction of pathogens by probiotics in cattle to the treatment of human disease,” Moon said.

He added that the circuit breaker’s reliability can lead to a wide range of potential applications down the line.

“The kill switch is generalizable. It’s not just for probiotics given to mice, but it can be grown into new bacteria. For example, I’m currently working on a similar switch, using bacteria to help the environment and solve the global plastic waste problem.

In its current project, supported by a grant from the Department of Energy, Moon hopes to turn plastic waste into profitable hybrid compounds. “Plastic bottles are everywhere right now. My hobby is collecting plastic bottles from campus that students forgot to put in a recycling bin and then using them as free materials,” he said. “We want to develop a process that uses plastic waste as an input and converts it into a high-value chemical, such as lycopene.”

Above all, Moon is passionate about helping people and hopes his life’s work will improve the health of individuals as well as the environment as a whole.

“I want to give people hope,” he said. “It’s been a crazy two years of COVID, and a lot of people are feeling hopeless. My life’s work is both in research and in helping others. I hope my story can encourage and inspire others.

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