Microbiome research has faced a hurdle from the get-go: the inability to edit the microbial genome in vivo. Until now, bacterial genomes had to be modified outside—and reintroduced into—the host animal. Now, that hurdle has been cleared. A group from Paris-based Eligo Bioscience has engineered a phage-derived vector to deliver a base editor and modify Escherichia coli while they are colonizing the mouse gut. It marks the first time the genomes of bacteria have been base edited, precisely and efficiently, directly in the gut.
The work, which is published online today in Nature in the paper “In situ targeted base editing of bacteria in the mouse gut,” was a team effort by the scientists at Eligo, led jointly by Jesus Fernandez-Rodriguez, PhD, (Eligo’s VP of Technology), David Bikard, PhD, (Eligo co-founder and head of the Synthetic Biology Group at Institute Pasteur), and Xavier Duportet, PhD, (Eligo’s CEO and chairman).
Before this report, notes Bikard, “it was still an outstanding question whether it would even be possible to genetically modify a whole target bacterial population in an animal. There could have been fundamental barriers that would’ve made this impossible. But here we show that we can do it!”
Eligo’s advance, which combined research in both vector engineering and payload modification, is exciting on two fronts: it could open the door to new microbiome genome-editing therapeutic modalities. And it launches a microbiome-editing toolbox that has been previously unavailable.
This work is “ushering in a new era of microbiome engineering,” notes Rodolphe Barrangou, PhD, professor at North Carolina State University and editor-in-chief of The CRISPR Journal. “This proof-of-concept study is just not for E. coli or the mouse gut microbiome; it can be used much more widely, for all kinds of things, and can be deployed at scale.”
Brady Cress, PhD, principal investigator of microbiome editing technologies at the Innovative Genomics Institute at the University of California, Berkeley, agrees. Cress told GEN that this is “a massive step forward that opens the door to rewriting our microbiomes for optimal health.”
Tweaking, not altering
Duportet co-founded Eligo with Bikard a decade ago; the two friends were still in training when they had the idea for the company—Duportet was a graduate student at MIT, Bikard a postdoctoral fellow in Luciano Marraffini PhD’s lab at the Rockefeller University. (Marraffini and Tim Lu, MD, PhD, are Eligo’s other scientific co-founders.) Today, Duportet and Bikard are a dynamic duo—with Duportet at the helm of the company and Bikard a scientific advisor, who remain close friends while collaborating scientifically.
Current microbiome approaches are typically based on altering the compositions of the bacteria. The idea is to introduce bacterial species to change the balance (like probiotics) or to remove others. Eligo’s focus here was different. The idea is not to kill the bacteria but rather, as Duportet explains, to “inactivate its pathogenic potential and leave the bacteria in place.”
“If you are trying to target bacteria that has a niche,” notes Bikard, “completely removing it from the niche might be very challenging. Unless there is something else there to take its place, it will just grow back. So, it is a better strategy to disarm it, rather than kill it.”
Two-fold
Eligo’s new data are not the first to demonstrate editing of the microbiome in vivo, however. In May 2023, research from the Danish company SNIPR Biome was published in Nature Biotechnology in a paper entitled, “Engineered phage with antibacterial CRISPR–Cas selectively reduce E. coli burden in mice.” In it, the researchers identified eight phages (after screening a library of 162 phages) that delivered a CRISPR-based gene-editing payload that resulted in a reduction of E. coli in the mouse gut. In the SNIPR Biome study, the E. coli were killed by CRISPR.
It has been known for a decade—since Bikard and Marrafini’s Nature Biotechnology paper in 2014—that cutting the bacterial chromosome with CRISPR-Cas kills bacteria efficiently. But the first generation of gene-editing tools were not very efficient tools, notes Bikard. They could be used in a lab setting to make modifications but most of the bacteria would be killed in the process. Therefore, if the goal is to make modifications in vivo, and maintain the bacterial population, the first generation of CRISPR tools for bacteria “would not cut it.”
The game changed when base editing—a more precise form of genome editing developed in the Broad Institute lab of David Liu, PhD—entered the toolbox. Eligo worked to bring together their knowledge in genome editing and delivery to allow for efficient editing of bacteria without changing the composition of the microbial population.
When targeting E. coli strains colonizing the mouse gut, Eligo’s technology modified the target gene in more than 90% of the bacteria, reaching up to 99.7% in some cases. These modifications remained stable for at least 42 days.
Barrangou notes that the penetrance of the edits showed remarkable efficiency. “They are setting the bar,” he told GEN. “Being able to do it is one thing. But being able to do it with that kind of longevity and efficiency is practically important and sets the stage for new opportunities in the field.”
For Bikard, working out the gene editing was not as big a hurdle as the delivery. Eligo modified the site fibers on a phage chassis (from phage lambda) to target specific bacterial strains. The phage vector can be modified to target different bacterial strains or species with additional engineering.
Cress thinks of it as “a reprogrammable platform” for targeting different bacteria. That said, while this research provides an impressive blueprint using the most well-studied phage-bacteria pair, Cress notes that expanding it to other microbes will necessitate developing efficient genetic tools for non-model bacteria and a deeper understanding of the genetics and biology of less well-studied phages.
Another advance, Duportet notes, is that his team was able to demonstrate the same efficiency of editing using a non-replicative plasmid in the target bacteria. This is an additional benefit because they don’t maintain a transgene in the microbiome of the animals.
Long road ahead
The long-term goal at Eligo is to develop therapeutics—not necessarily for infectious diseases. The interest extends to those that would change the genetic content of the microbiome that alter a factor of host–focused diseases.
One example where this could be applicable is the delivery of a base editor to commensal intestinal E. coli that express the toxin colibactin, to inactive its mutagenic potential, therefore preventing the progression of human colorectal tumors.
But there is a long road ahead and challenges remain. One, notes Cress, is that this approach uses short-lived delivery of gene editing machinery to make gene disruptions, but other types of edits like gene insertions take longer to write (e.g. CRISPR-associated transposases) and thus will likely require different delivery approaches. Another point Cress considers is that “the type of edits made in this study could potentially revert through natural mutation, making gene removal a more durable solution than gene disruption.”
This study also raises new questions. Teasing apart the genetic network of the microbiome is in its infancy. Do researchers have enough knowledge to use this new tool?
“It’s important to have the genome editing tools,” notes Barrangou. But in the end, “what really matters is knowing what to target. Knowing what to target and what edit you want is part of the secret sauce.”
But Bikard reckons that this work will help answer some of those questions. This will be an extraordinary tool for researchers, he says, because it offers the possibility to probe gene function directly in the animal. He is excited to use it in his academic lab on the other side of Paris from Eligo’s base.
Duportet hopes that scientists will use the method and is happy to issue “a call for collaboration.” “We cannot work on everything, and we cannot find all the targets to edit,” he notes. “But we have the knowledge to design the vectors and the payloads to make it happen.”
