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These 3D-printed extracellular vesicle (EV) droplets enable DNA and RNA sequencing to differentiate extracellular vesicle subpopulations. Extracellular vesicles are nanosized vesicles containing a variety of cargo, including mRNA, proteins, and lipids, that nearly all cell types release. EVs are essential to many physiological processes, such as coagulation, inflammatory response, cell maturation, adaptive immune response, bone calcification, and neural cell communication. They also play fundamental roles during cancer progression, suppressing immune surveillance in the tumor microenvironment and establishing the premetastatic niche. Due to the EVs’ ability to transfer their contents to cells in the body and their stability in the bloodstream, there is broad translational potential for EVs in oncology, such as tumor biomarkers. However, the current single-vesicle analysis uses bulk approaches, creating heterogenous EV samples in vesicle structure, contents, and function. This heterogeneity impedes EV development as a biomarker.
Another issue is current microfluid technologies. These technologies form droplets once a single cell enters a fluidic orifice, creating “drops on demand” containing single cells. Current microfluidic technologies at the nanoscale are carefully controlled and are susceptible to low throughput and clogging. Current systems cannot detect EVs beyond a certain threshold; thus, creating single-cell EV droplets is not feasible.
Researchers at the University of Florida have developed 3D-printed aqueous droplets containing extracellular vesicles (EVs) for sequencing the DNA and RNA of single-cell EVs. DNA/RNA sequencing of an individual EV can identify its nucleic acid contents, including structure, composition, and contents for performing a specific function. By characterizing an EV at a single-vesicle resolution level, EV subpopulations of composition and content are differentiated from a heterogenous sample, closing the gap to EVs’ potential as a biomarker. The global EV market was estimated to be worth $169M in 2023 and is poised to reach $356 M by 2028, growing at a CAGR of 16% from 2023-20281.
DNA and RNA sequencing of individual extracellular vesicles (EVs) via 3D printing the EVs within aqueous droplets, differentiating EV subpopulations
This technology uses a 3D printing apparatus to form an aqueous droplet containing a single extracellular vesicle (EV) and a single DNA-barcoded microbead, enabling DNA/RNA sequencing within each EV. A drop of aqueous solution contains an EV and microbead for performing biochemical reactions. The droplet is 3D printed on a medium via a nozzle connected to a supply of the aqueous solution the droplets are made of. Once on the medium, the biochemical reaction occurs within the droplet, and the droplets are separated or pooled from the medium via centrifugation. The aqueous phase with the droplet contents is then removed from the medium. This technology addresses EV heterogeneity and enables the composition, contents, and function identification of extracellular vesicle subpopulations.
This gene editing of the gut microbiota provides a way to study the role bacterial genes play in disease and health. The microbiota is considered an important modulator of human health and disease. The intestinal, or gut, microbiota, is the richest and most complex microbiota ecosystem in the body. It provides many beneficial functions such as educating our immune systems, synthesizing essential vitamins and nutrients, and fending off invading pathogens. However, the gut microbiota is sensitive to environmental changes such as medications, stress, or diet. Studies have shown these changes to the microbiota environment could promote diseases such as digestive disease, arthritis, asthma, cardiovascular disease, neurological disorders and cancer. Scientists are unable to study the different microbiota genes functionally linked to these diseases because more than 90% of the bacteria forming in the microbiota are not genetically amendable by current gene editing techniques, such as flp recombinase, transposon, or chemical screen. This poses a severe obstacle in scientists’ abilities to conduct mechanistic studies of microbiota function in disease, preventing the development of therapeutic treatments. New tools must be employed to alter bacterial genomes to open the way for novel therapeutics.
Researchers at the University of Florida can manipulate or silence specific gut microbiota through exosome-mediated siRNA. Scientists around the world will have the ability to design functional experiments targeting specific genes. They will be able to provide critical information about the role of microbiota in various biological processes and diseases, opening the way for therapeutics using precision medicine for microbiota.
Mechanism for specific gene silencing in the gut microbiota, allowing for the study of the role bacterial genes have on human health and disease
This gene editing system utilizes a transkingdom communication network between mammals and bacteria. The mammalian sRNAs are processed and loaded into the RNA Induced Silencing Complex (RISC). Gene expression is either silenced (siRNA) or repressed (miRNA) following the recognition of the sRNA to its’ target. These are then packaged into an exosome, loaded with the RNA Induced Silencing Complex, and purified, before being added to a bacterial genome to silence specific genes. In addition, the RNA Induced Silencing Complex can be programmed to target a specific gene before being introduced in the bacteria.
