Modifying the Microbiome In Vivo, One Species or Gene at a Time

Over the past 25 years, researchers have learned a lot about our microbial companions and generated a mountain of data thanks to advances in sequencing technology. But researchers still lack effective methods to alter the microbiome based off of this information. Introducing new microbes via supplements or fecal transplants has shown little success because new bacterial residents have a hard time grabbing a foothold in a crowded, established community. Killing pathogens with antibiotics also takes benign bacteria as collateral damage, and can lead to drug resistance in survivors.

Researchers target E. coli in the mouse gut with a CRISPR system 

that induces DNA breaks at a specific genomic sequence.

“There's a lack of precision in terms of how we are able to manipulate the microbiome,” said Peter Turnbaugh, associate professor at the University of California, San Francisco, whose research team recently developed a microbiome-altering system using CRISPR-Cas9 editing technology. “That really motivated this sort of search for a tool that we could use to make more specific changes.”

In vitro, scientists remove genes or kill cells by inducing double-strand DNA breaks at targeted sequences with CRISPR’s Cas nuclease.  As described in a recent Cell Reports study¸¹ Turnbaugh’s team developed a precise way to modify the microbiome in vivo with CRISPR, one bacterial strain or gene at a time.

“The approach is definitely very interesting, and…the field is trying to go to towards specific, surgical modifications of the microbiome,” said Michael Zimmermann, a group leader at the European Molecular Biology Laboratory, who was not involved in this study. “It's pretty clear that the microbiome has this link to health and disease and we need to modulate it.”

Special Delivery

The trick to using CRISPR to change the microbiome of an organism was to find a way to deliver it to the bacteria of interest—in this case, the mouse gut microbiota. To accomplish this, Turnbaugh turned to bacteriophages, which have a special relationship with the bacteria they infect; most phages only infect one bacterial species or strain. While some kill a cell upon infection, filamentous phages, such as E. coli’s M13 used in this study, take a gentler approach and can be co-opted to deliver genetic cargo into their host. The researchers decided to package the phages with plasmids containing the CRISPR system engineered to target a specific DNA sequence.

To visualize CRISPR at work, the scientists designed the system to disrupt a bacterial gene locus encoding green fluorescent protein (GFP). Prior to phage treatment, the scientists colonized mice with a GFP E. coli strain and a red-fluorescent (mCherry) strain as a control. In later experiments, the researchers used a single bacterial strain that contained both fluorescence genes. Next, they orally administered the phages to the mice, sending them to interact with E. coli in the gut. Turnbaugh’s team expected the CRISPR system to cut the bacterial genome at the GFP locus, which would kill most cells unable to repair the damage.

To determine their experiment’s success, the researchers isolated bacteria from mouse stool samples and analyzed fluorescence. Overall, the CRISPR-carrying bacteriophage killed most of the GFP-containing bacteria in the mouse gut. In the surviving bacteria collected from the stool, the researchers observed many red fluorescent cells and few green and non-fluorescent E. coli. 

Whole-genome sequencing indicated that the surviving phage-infected bacteria that lost their green glow had large deletions in the GFP gene, which came from imprecise repair of Cas9-induced double-strand DNA breaks. This showed that for the most part, the phages found their marks and delivered the CRISPR cargo, which targeted and cleaved the intended sequence.

Improving for the Future

Turnbaugh sees this work as a tool for researchers to use in the laboratory. By precisely editing bacterial genomes in vivo, they can understand important mechanisms driving health and disease associated with the microbiome and pathogenic microbes.

Turnbaugh’s team is currently resolving the system’s limitations. Notably, the mouse model does not mimic the human gut—the researchers treated the mice with antibiotics for several reasons, including to wipe out the gut microbiome to make room for the fluorescent E. coli.  Additionally, according to Zimmermann, another limitation is that the only bacteria tested were E. coli—a concern that Turnbaugh hopes to address in the future.          

The filamentous bacteriophage M13 (yellow) injects a plasmid carrying

 sequences for the CRISPR machinery into E. coli (blue).

“We have tons of data from around the world looking at the diversity of bacteriophage in the gut, but what we don't have are really well-characterized pairs of bacteria and viruses, like M13 and E. coli,” said Turnbaugh. “One of the major things moving forward is identifying other pairs. We'd like to have more phage that would target other members of the community, ideally in a way that's not really strain specific.”

Additionally, the system is not foolproof because the researchers saw bacteria in stool that avoided CRISPR attack. “Bacteria are the original, incredible escape artists. There are ways that they have of evading whatever you're trying to do,” said Kathy Lam, the paper’s first author. “You're making a double-stranded cut. It's not weird that they can repair that in ways that allow them to survive, or to mutate the CRISPR-Cas system.”

Evidence of bacteria repairing their DNA led the researchers to see this system less as an antimicrobial designed to kill bacteria, but as a way to understand and modulate pathogens or the microbiota by disrupting targeted genes. “If you have organisms that are in your gut, and they're breaking down molecules, it's important to understand those activities. And if they're undesirable, you could potentially remove this specific gene that is performing that activity,” Lam said.

Beyond removing unwanted bacteria or genes, this phage delivery system could deliver desirable genes to microbiota residents, which opens many possibilities. “The alternative strategy that we're thinking about is…gaining function,” said Turnbaugh. “Ideally, we want to add functions to E. coli that are both useful to the bacteria and relevant to disease…We want to try to work with E. coli to make a healthier gut.”