Billions of bacteria, fungi, and other microbes live throughout our bodies — collectively known as our microbiome. These microscopic communities often coexist peacefully with human cells, even helping carry out key bodily processes. But when they go rogue, they can promote conditions ranging from Crohn’s disease to acne.

Genome editing can provide a new toolkit to treat these diseases — but not by editing human cells. Instead, scientists are now using molecular scalpels like CRISPR to edit the genomes of microbes that wreak havoc within the human body.

“It is ironic that even though genome-editing tools like CRISPR-Cas9 were invented by bacteria, they’ve been used most efficiently in mammalian cells,” said Peter Turnbaugh, a microbiologist at the University of California, San Francisco. “They haven't had the same kind of transformative effect on bacterial genetics as they have on human genetics.”

Returning these tools to their bacterial roots is no small task, though. It requires coming up with new ways of delivering genome-editing machinery to cells hidden deep in the recesses of human intestines or under layers of mucus. This has been the focus of recently formed companies such as Eligo Bioscience, a French company focused on editing the human microbiome in vivo. They and others are drawing inspiration from bacteria-infecting viruses called phages that might offer important lessons for how to target wayward microbes.

Maintaining a delicate balance

The human body is coated in an invisible living layer of microbes stationed along the walls of the intestines, the surface of the skin, and in the crevices between teeth. There, they play a role in numerous tasks in digestion, immunity, and other essential human life processes. For example, bacteria in the gut help ferment dietary fiber. Microbes in the airways fend off other potentially harmful bacterial infections.

Alterations to these bacteria’s levels can have big implications for human health, so the microbiome has long been an intriguing target for drug development. The FDA approved the first gut microbiome-based drug, called SER-109, in 2023 (1). This pill contains bacteria that can fight dangerous Clostridioides difficile infections in the intestines. Another example is topical antifungals, which can treat skin conditions such as dermatitis caused by microbial imbalance.

But not all microbes are inherently harmful. Many of the species on the human body are commensal or even mutualistic: They benefit from living on the human body, and we benefit from the functions they carry out. These may become temporarily dysfunctional, but their eradication may be ultimately harmful. This has been a growing concern with antibiotics, for example, which can wipe out good bacteria in the human body in the pursuit of killing a pathogen.

“If you get sick and you take a drug for your liver, you're not trying to remove your liver,” Turnbaugh said. “Similarly, we really want to be able to maintain our long-term relationship with these commensal microbes.”

Enter phages: viruses that infect bacteria. They’re often depicted to look like a futuristic space probe, with angular, spidery legs and icosahedral heads. Some estimates suggest there are ten times more phages than bacteria in the world. “A lot of people call them the dark matter of the gut microbiome,” said Bryan Hsu, a microbiologist at Virginia Tech.

Phages have the unique ability to target a specific bacterial strain, attach to its surface, and inject genomic material into the bacterium. They can then hijack the bacterium’s processes to repurpose it as a phage-making factory. This makes phages an intriguing delivery mechanism for microbe-targeting therapies.

Scientists have tried to use phages therapeutically before. The category of therapies called “phage therapies” uses lytic phages to kill cells — although none of these therapies are FDA approved yet and are only used as a last resort for particularly devastating microbe-related illnesses.

But what if killing a cell isn’t the goal? “You have such a competitive environment in the gut microbiome that other bacteria would love to take their place,” Hsu said. Or worse, a single surviving member of the eradicated species could spawn more bacteria that share whatever genes made their predecessor resistant to the phage — similar to the emergence of antibiotic resistance.

Instead, using phages to deliver gene-editing machinery allows scientists to modulate the functions of microbes without killing them. If a microbe is producing a disease-causing molecule, the CRISPR-Cas9 system can precisely inactivate that gene without killing the whole bacterial population.

Although CRISPR systems have been widely used for more than a decade, adapting them to bacterial cells poses some hurdles. For example, bacterial cells are very sensitive to double-stranded DNA breaks, so researchers must ensure that snipping the DNA doesn’t lead to toxic responses that kill the bacteria. “It makes it much harder to just remove a gene without affecting the level of the cell in the population,” Turnbaugh said. In a 2021 proof-of-concept study published in Cell Reports, Turnbaugh’s lab used an engineered phage to inactivate a gene in Escherichia coli  in a mouse’s gut (2).

Phages can also deliver variations of the CRISPR-Cas9 system to accomplish other types of genomic edits in the microbial cells. For example, in a 2020 study, Hsu demonstrated that a temperate phage — which integrates its genomic material into bacteria, rather than killing them — could deliver “dead Cas9” to transcriptionally repress a gene (3). This inactivated Cas9 enzyme binds to a gene but rather than cutting it, it physically blocks the gene’s transcription into RNA, preventing its protein production.

A strategy like this could be useful for fixing normally benign bacteria that are producing a toxin, Hsu said. One example he has targeted is Shiga toxin in E. coli, which is the root of many foodborne illnesses (4).

Drawing inspiration from phages

For Xavier Duportet, the most important question is not what genome-editing machinery to deliver, but how to deliver it. He became interested in this topic over a decade ago when he was a graduate student at the Massachusetts Institute of Technology. He collaborated with a friend at Rockefeller University on a study in which they showed that they could use Cas9 to target virulence and antibiotic genes in Staphylococcus aureus (5). At the time, they used phages as their vector.

Now, as a cofounder and Chief Executive Officer of Eligo Bioscience, Duportet no longer thinks phages are the solution, but he still draws inspiration from them. After all, “this is the most precise antimicrobial you can find because it can discriminate strains of E. coli just because they have a two base-pair difference in their genome,” he said. But phages come with baggage: Their goal is to replicate in their target bacteria, and this triggers anti-viral defenses.

Instead, Eligo Bioscience is developing a delivery platform that uses phage-derived particles instead of phages themselves. The particles consist of just the capsid — the structure that normally encapsulates the phage’s genome — minus any of the phage’s actual genetic material. The team has engineered the capsid to maintain the proteins that phages use to recognize bacterial strains, and they can customize the proteins to the phage’s intended target.

Hsu was impressed by the way the researchers at Eligo Bioscience engineered their phage-like particles, especially to keep them from replicating and expanding their range of targets. “Alone, some of these ideas have been out there, but putting them together into a phage is quite interesting,” Hsu said.

Once they build this capsule, they can fill it with DNA encoding whatever genomic material they want to inject into the cell. The company is currently focused on three possible applications. The first is exactly what Duportet worked on as a graduate student: delivering gene-editing machinery to create lethal DNA breaks that kill a target strain. This creates an alternative form of an antibiotic but with high specificity to a single strain, Duportet noted. The second application is to introduce pieces of genomic material into bacteria to be temporarily expressed.

In this complex ecosystem, if you try to kill something that should be there, you're not going to be able to kill it. It's going to come back.
- Xavier Duportet, Eligo Bioscience

The final mechanism, which has attracted the most attention, is to use high-precision gene-editing machinery to modify the bacterial genome to either stop producing a toxin or to start producing a therapeutic molecule. To do so, though, Duportet needed more than just the coarse cuts that CRISPR-Cas9 can make.

In a 2024 study published in Nature, Eligo Bioscience scientists demonstrated that they could use phage-derived particles to deliver base editors into gut microbes in a mouse (6). Base editors are a newer gene-editing technology that can target and change individual nucleotides. Duportet believes this technology will drastically expand the kinds of modifications that researchers can introduce into the microbiome. For example, he imagines editing proteins that cause autoimmunity so that the human immune system no longer responds to them or editing cancer-causing proteins to inactivate them.

Both Hsu and Turnbaugh, who were not involved in the study, noted how effective the genome editing was: The base editors achieved a median of 93 percent editing efficiency. Once the edits are in the genome, the bacteria can pass them down to their offspring. “If you don't create a fitness disadvantage in the bacteria, the edit can last forever,” Duportet said.

Hsu was also impressed by how well the phage-derived particles reached their target in a mouse model. Targeting gut microbes poses additional challenges for delivery because the phage-derived particle must endure a gauntlet of hostile environments and find the bacteria hiding in the gut, Hsu added. “In the gut, you have a ton of different bacteria all over the place, sometimes just in certain compartments, or they're hiding in the mucosal lining a lot of the time,” he said.

Good performance in the gut is important for Eligo Bioscience because they are working on disrupting intestinal E. coli production of Shiga toxin to treat gastrointestinal diseases caused by the pathogen. In this case, Duportet thinks that modifying the bacterial function is a better strategy than killing the E. coli, which is a normal part of the gut microbiome.

“It's nature,” he said. “In this complex ecosystem, if you try to kill something that should be there, you're not going to be able to kill it. It's going to come back.”

The next generation

Although gut microbes are often the focus of microbiome research, Eligo Bioscience’s priorities lie elsewhere. Their first target is actually a very common skin condition: acne. Skin is covered in bacteria, much of which are harmless or even helpful. Skin bacteria that produce pro-inflammatory molecules, such as Cutibacterium acnes, however, can cause pimples. These bacteria can lead to dozens of skin lesions and scarring that are hard to tamp down.

Duportet was underwhelmed by the currently available acne therapies. “It has been 30 years, but there's no real innovation in the acne field,” he said.

Traditional antimicrobial therapies would kill both the good and the bad bacteria, Duportet said, so Eligo Bioscience is developing a topical gel that will deliver phages to destroy the bad bacteria by targeting their inflammatory genes. They plan to start clinical trials in the United States by late 2025 or early 2026.

Even as Eligo Bioscience charges ahead to develop this new therapy, there are still many basic questions about microbiome gene editing that researchers are investigating. Turnbaugh, for example, wants to understand how bacteria might evolve strategies to avoid these therapies. In his 2021 study, he found that some bacteria removed or inactivated the CRISPR machinery that the phage introduced. “What we mainly uncovered were lots of ways that this could be improved upon in the future to avoid routes of escape from genome editing,” Turnbaugh said.

Hsu is optimistic about the future of the technology, especially after seeing gene-editing research groups such as Jennifer Doudna’s Innovative Genomics Institute turn their attention to microbiome editing. Doudna’s team has also been developing gene-editing tools for the microbiome for a few years now, and they plan to supercharge this work with a recent $70 million grant (7). One project that has emerged from this is an effort to edit the genomes of asthma-related microbes as a new approach to treat the disease.

“I think that we're starting to get to the next generation of what people consider therapeutic applications of phages,” Hsu said.

References

1. Feuerstadt, P. et al. SER-109, an Oral Microbiome Therapy for Recurrent Clostridioides difficile Infection. N Engl J Med 386, 220-229 (2022).
2. Lam, K.N. et al. Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome. Cell Rep 37, 109930 (2021).
3. Hsu, B.B. et al. In situ reprogramming of gut bacteria by oral delivery. Nat Commun 11, 5030 (2020).
4. Hsu, B.B. et al. Stable Neutralization of a Virulence Factor in Bacteria Using Temperate Phage in the Mammalian Gut. mSystems 5, e00013-20 (2020).
5. Bikard, D. et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol 32, 1146-1150 (2014).
6. Brödel, A.K. et al. In situ targeted base editing of bacteria in the mouse gut. Nature 632, 877-884 (2024).
7. Rubin, B.E. et al. Species- and site-specific genome editing in complex bacterial communities. Nat Microbiol  7, 34-47 (2022).