Editing Specific Bacteria in Soil Microbiomes Using Phage Delivered Base Editors

Scientists at North Carolina State University have developed a method to precisely edit a single gene in a targeted bacterial species within a microbial community, using viruses that attack bacteria (bacteriophages). The findings were published in the journal Proceedings of the National Academy of Sciences.

“We see this as a mechanism to aid the microbiome. We can make a change to a particular bacterium and the rest of the microbiome remains unscathed,” said Rodolphe Barrangou, PhD, distinguished professor of food, bioprocessing, and nutrition at North Carolina State University. “This is a proof of concept that could be employed in any complex microbial community, which could translate into better plant health and better gastrointestinal tract health—environments of importance to food and health.”

First, the authors investigated whether the bacteriophages T7 and λ could be engineered to deliver the CRISPR machinery to an E. coli bacterium. Phages such as λ can integrate their genetic material into the genetic material of a host bacterium and replicate in concert with it. Such a viral life cycle is called “lysogeny.” In the current study, the investigators noted, λ lysogeny is effective in expressing extraneous base editors in E. coli to knock out specific genes. They showed, both engineered bacteriophages—T7 and λ—delivered payloads to the E. coli host. These payloads expressed bacterial fluorescent genes and manipulated the bacterium’s resistance to an antibiotic.

The researchers used engineered bacteriophages to deliver the CRISPR-Cas editing machinery both in vitro in test tubes and in situ where the target bacteria exist within a microbial community. To develop a model microbial community that includes a range of bacteria, the team used an EcoFAB device filled with sterile white quartz sand to act as synthetic soil.

Understanding interactions in the soil rhizosphere—the narrow layer of soil that is affected by secretions from roots and associated microorganisms—is important to ensure the sustainability of food resources. This study establishes the feasibility of manipulating soil microbial communities to control the composition and function of bacteria associated with plants. The findings could help design ecosystems to promote the growth and health of plants, which is crucial for sustainable agriculture.

The approach developed in the study is ironic since biologically, bacteria use CRISPR-Cas systems to counter attacking viruses. Biotechnology has adapted CRISPR-Cas systems to edit genes in animals and humans. But in the current study, the researchers flip the board and load viruses with CRISPR-Cas machinery to change bacterial behavior, specifically when the target bacterium exists within a microbiome.

“Viruses are very good at delivering payloads. Here, we use a bacterial virus, a bacteriophage, to deliver CRISPR to bacteria, which is ironic because bacteria normally use CRISPR to kill viruses,” said Barrangou. “The virus in this case targets E. coli by delivering DNA to it. It’s like using a virus as a syringe.”

The researchers used λ phage to deliver a cytosine base editor (CBE) into E. coli which changed a single base in E. coli’s DNA with sensitivity and precision.

Lead author of the study, Matthew Nethery, PhD, said, “We used a base editor here as a kind of programmable on-off switch for genes in E. coli. Using a system like this, we can make highly precise single-letter changes to the genome without the double-strand DNA breakage commonly associated with CRISPR-Cas targeting.”

In EcoFab’s fabricated ecosystem, the researchers included three different types of bacteria to investigate if the phage could specifically locate E. coli within the system. The engineered λ phage was efficient in attaching to E. coli and edited its genome.

“In a lab, scientists can oversimplify things,” said Barrangou. “It’s preferable to model environments, so rather than soup in a test tube, we wanted to examine real-life environments.”

Trent Northen, PhD, a scientist at the Department of Energy’s Lawrence Berkeley National Laboratory who collaborates with Barrangou’s team said, “This technology is going to enable our team and others to discover the genetic basis of key bacterial interactions with plants and other microbes within highly controlled laboratory environments such as EcoFABs.”

Barrangou’s team intends to test the phage CRISPR technique with other soil-associated bacteria, in future experiments.

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