application-flow-vermimetry-for-quantification-analysis

Application of Flow Vermimetry for Quantification and Analysis of the <em>Caenorhabditis elegans</em> Gut Microbiome

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring ...

Baylor College of Medicine

The free-living nematode Caenorhabditis elegans remains one of the most robust and flexible genetic systems for interrogating the complexities of animal biology. Targeted genetic manipulations, such as RNA interference (RNAi), CRISPR/Cas9- or array-based transgenesis, all depend on initial delivery of nucleic acids. Delivery of dsRNA by feeding can be effective, but the expression in Escherichia coli is not conducive to experiments intended to remain sterile or with defined microbial communities. Soaking-based delivery requires prolonged exposure of animals to high-material concentrations without a food source and is of limited throughput. Last, microinjection of individual animals can precisely deliver materials to animals' germlines, but is limited by the need to target and inject each animal one-by-one. Thus, we sought to address some of these challenges in nucleic acid delivery by developing a population-scale delivery method. We demonstrate efficient electroporation-mediated delivery of dsRNA throughout the worm and effective RNAi-based silencing, including in the germline. Finally, we show that guide RNA delivered by electroporation can be utilized by transgenic Cas9 expressing worms for population-scale genetic targeting. Together, these methods expand the scale and scope of genetic methodologies that can be applied to the C. elegans system.

Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation.

The microbiome is increasingly receiving attention as an important modulator of host health and disease. However, while numerous mechanisms through which the microbiome influences its host have been identified, there is still a lack of approaches that allow to specifically modulate the abundance of individual microbes or microbial functions of interest. Moreover, current approaches for microbiome manipulation such as fecal transfers often entail a non-specific transfer of entire microbial communities with potentially unwanted side effects. To overcome this limitation, we here propose the concept of precision prebiotics that specifically modulate the abundance of a microbiome member species of interest. In a first step, we show that defining precision prebiotics by compounds that are only taken up by the target species but no other species in a community is usually not possible due to overlapping metabolic niches. Subsequently, we present a metabolic modeling network framework that allows us to define precision prebiotics for a two-member C. elegans microbiome model community comprising the immune-protective Pseudomonas lurida MYb11 and the persistent colonizer Ochrobactrum vermis MYb71. Thus, we predicted compounds that specifically boost the abundance of the host-beneficial MYb11, four of which were experimentally validated in vitro (L-serine, L-threonine, D-mannitol, and γ-aminobutyric acid). L-serine was further assessed in vivo, leading to an increase in MYb11 abundance also in the worm host. Overall, our findings demonstrate that constraint-based metabolic modeling is an effective tool for the design of precision prebiotics as an important cornerstone for future microbiome-targeted therapies.

Article	Year
Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation. G3 (Bethesda, Md.)\| PubMed ID: 33872353	2021
Metabolic model predictions enable targeted microbiome manipulation through precision prebiotics. bioRxiv : the preprint server for biology\| PubMed ID: 36824941	2023

Dana Blackburn

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