Reshaping the Gut Microbiome Using New Genetic Tools

Jan. 4, 2021

To understand the biological underpinnings of a complex system like our gut microbiota, we need tools that can perturb it. Currently, many studies on how the microbiome impacts our health are associative and descriptive: they lack mechanistic insight into microbe-microbe and microbe-host interactions. One reason for this is that gut bacterial species are not easy to culture in the lab, making the development of gene editing tools for them extremely challenging. However, deleting specific genes from specific strains in the gut microbiota could help assign function to them, and in turn determine how such functions influence our health. Several groups have recently developed promising gene editing tools for individual species of gut bacteria or entire bacterial communities. These tools could not only help the gut microbiome field advance from association to causation, but could form the basis of future therapeutics.

Innovation Strategies for Genetically Intractable Bacteria

Bacteroidales, which encompasses the most abundant gut genera (i.e., Bacteroides, Parabacteroides and Prevotella), are naturally resistant to a wide variety of antibiotics that are used for genetic selection in the lab, making their manipulation challenging. In 2015, Mimee et al. developed a toolbox of promoters, ribosome-binding sites and inducible systems for the regulation of gene expression in the commensal Bacteroides thetaiotamicron, one of the most widely-studied gut bacteria with known immunomodulatory functions. Using this toolbox, the authors developed an inducible CRISPR interference (CRISPRi) platform using guide RNA and a catalytically inactive Cas9 to repress specific genes by blocking their transcription by RNA polymerase. The authors used their toolbox and CRISPRi platform to implement complex gene circuits in B. thetaiotmicron, both in vitro and in mice. Interestingly, by supplementing the drinking water of mice with specific inducers like arabinose or rhamnose, the authors were able to turn on the respective genetic circuits they engineered in B. thetaiotamicron in the context of a complex microbiota. 
Scientists have also developed new ways to easily replace alleles in diverse gut Bacteroidales via 2-step allelic exchange. Garcia-Bayona and Comstock developed vectors that use the ability to use a plant polysaccharide for positive selection rather than resistance to an antibiotic. Their study, and that of Bencivenga-Barry et al., also developed a negative selection system based on an inducible type VI secretion system toxin. These vectors have significantly reduced the time and labor of gene editing in Bacteroidales.

Inulin selection and rhamnose counter-selection plasmids.
Top left, inulin positive selection plasmid integrates at the chromosomal attBT2 sites. Top right, a plasmid with tetracycline positive selection and rhamnose-inducible ssBfe1 counterselection. Bfe1 is a highly toxic effector from B. fragilis type VI secretion system. Bottom, vector combining the inulin positive selection cassette and rhamnose-inducible ssBfe1 counterselection cassette.
Clostridia, another abundant gut bacterial class, produce many metabolites that diffuse into the host’s circulation. Clostridia are not readily amenable to efficient genetic manipulation, but Guo et al. recently developed a CRISPR-Cas9 gene knockout system in Clostridium sporogenes that demonstrated a potential link between branched short-chain fatty acids produced by C. sporogenes and host immunoglobulin A response. Another study by Canadas et al. demonstrated the first use of synthetic riboswitches to significantly increase the efficiency of CRISPR-based editing. Riboswitches are mRNA elements most commonly found in 5’-untranslated regions that interact with metabolites to regulate the expression of the coding region. These authors applied their method (called RiboCas) in 4 Clostridia, with potential to expand to other Clostridia and genera, as well.
CRISPR-Cas9-based gene editing tools have also been developed in probiotic Lactobacillus species, including Lactobacillus reuteri and Lactobacillus plantarum. Engineering probiotics could aid in developing strategies to enhance their survival in the gut and to study their immunomodulatory properties, as many Bifidobacteria and Lactobacilli interact with the host immune system via surface-associated proteins. 
Another complementary technique is a top-down approach that relies on rewriting the metagenome of the entire gut community instead of targeting specific microbes. In January 2019, the Wang Lab at Columbia University developed one such platform called Metagenomic Alteration of Gut Microbiome by In situ Conjugation (MAGIC) to genetically manipulate complex microbial communities in their native habitat. Bacteria notoriously swap their genes via conjugation. So, donor MAGIC Escherichia coli strains can deliver engineered genes to a broad range of bacterial recipients. Using a conjugation system that can deliver plasmids to both gram-positive and gram-negative bacteria in a mouse model, Ronda et al. were able to modify genomes of 297 gut bacterial species (~5% of the microbiome) across 4 phyla. However, the transconjugants were no longer detected after 3 days in situ, suggesting future work must improve the lifespan of the payload. 

Therapeutic Applications of Genetically Engineered Gut Microbiota 

Comparisons of the gut microbiome in diseased and healthy hosts have led to several microbiome-based therapeutics including probiotics, prebiotics and fecal microbiota transplants (FMTs). In particular, FMTs, which rely on the transfer of the entire microbiota from a healthy to an ill person, have demonstrated success in treating recurring and refractory Clostridium difficile infections. But, it is difficult to standardize this method and gauge its long-term consequences. On March 12, 2020, The U.S. Food and Drug Administration (FDA) issued a safety alert regarding infections caused by drug-resistant bacteria following FMT in 6 patients, resulting in 2 deaths and 4 hospitalizations. Developing tools for precise engineering of the microbiota (either individual strains or the whole community) can circumvent these challenges by reducing the risk of inadvertently transferring unknown organisms to a patient.

Scientists have already engineered commensal strains of E. coli to produce therapeutic molecules that can treat metabolic deficiencies, inhibit tumor growth and clear infections by pathogens in mice, as well as present essential antigens to the immune system for vaccine development. In 2019, Zbiotics started the sale of the world’s first genetically modified probiotic, a Bacillus subtilis strain engineered to break down acetaldehyde, a molecule thought to be the cause of hangover headaches. However, none of their studies provide any evidence that the engineered bacteria breaks down acetaldehyde in humans or animals. In fact, lack of proper clinical trials demonstrating efficacy of engineered probiotics is a major source of public skepticism. Hence, understanding their mechanism of action via genetic engineering and/or other biological tools is critical. 
So, what is in store for engineered gut bacteria for therapy?  As with any new therapeutic technique, there are always challenges associated with making the leap from test tubes, to animal models, to clinical trials. It will be imperative to ensure that engineered commensals are safe and don’t share their DNA with other bacteria in the environment. A possible way around the latter is to engineer a failsafe in genetically modified bacteria that would trigger bacterial death in desired conditions, for instance, after they have served their therapeutic function. 
In spite of these hurdles, the potential payoffs of developing methods to modify gut microbes, either individually or as a community, to advance human health are immense. Ultimately, these methods will lay the groundwork and experimental setup for the microbiome field to move beyond correlations and establish causations in microbe-microbe and microbe-host interactions. One day, we may be able to create a synthetic microbiome with bacterial species engineered to work together to promote health by producing beneficial molecules.


Author: Kanika Khanna, Ph.D.

Kanika Khanna, Ph.D.
Kanika Khanna, Ph.D., is a postdoctoral scholar at the University of California, Berkeley studying the structural basis of membrane manipulation and cell-cell fusion by bacterial pathogens.