Genetically engineered bacteria from the microbiome of bees are used to produce RNA interference (RNAi) molecules and other genetic tools to target and downregulate specific genes in bees or their pathogens. By reducing pathogen loads and altering gene expression of bees, this technology can combat threats to bee population, like colony collapse disorder, and improve bee health.
The decline in bee populations, particularly honeybees, poses a significant threat to global agriculture due to their critical role in pollination. This decline is attributed to various factors, including pathogens, parasites like the Varroa mite, and environmental stressors such as pesticides. Traditional methods to combat these issues, such as chemical treatments and breeding for resistance, have limitations including high costs, environmental impact, and the development of resistance.
RNA interference (RNAi) has emerged as a promising technique to specifically target and downregulate harmful pathogens and parasites through their genes. However, current RNAi delivery methods, such as direct injection or feeding of double-stranded RNA (dsRNA), are impractical for large-scale application due to their instability and the trauma they cause to bees. The production of dsRNA is also expensive, and its effectiveness is often compromised by rapid degradation in the environment. There is a need for a more efficient, sustainable, and scalable approach to deliver RNAi in bee populations to enhance their resilience against pathogens and environmental stressors.
Genetically engineered bacteria, native to the microbiome of insects like bees, are utilized to induce RNA interference (RNAi) by expressing heterologous nucleic acids, including double-stranded RNA (dsRNA). These bacteria are transformed using a broad-host-range plasmid from the RSF1010 replicon, which supports various genetic constructs such as antibiotic resistance markers, promoters, and fluorescent reporters. The plasmids are assembled using Golden Gate cloning, allowing modular and combinatorial assembly of genetic parts.
Once introduced into the insect’s gut, the engineered bacteria colonize and produce dsRNA, leading to RNAi effects that can reduce pathogen load or alter host gene expression. The toolkit also includes components for CRISPR interference (CRISPRi) and Cas9-assisted gene disruption, enabling targeted gene repression and knockout in the bacterial chromosome. This approach can study microbiome interactions and develop biotechnological applications to improve insect health and mitigate threats like colony collapse disorder in bees.
This technology is differentiated by its use of a broad-host-range plasmid, RSF1010, which allows it to function across diverse bacterial species. The RSF1010 origin is known for its ability to replicate in various bacterial lineages, including Cyanobacteria and Agrobacterium. The modular nature of the Golden Gate cloning system further enhances its adaptability, allowing researchers to test different genetic constructs quickly. The inclusion of CRISPRi and Cas9 components for targeted gene manipulation adds another layer of precision and functionality. This combination of broad-host-range compatibility, modular assembly, and advanced genetic tools makes this technology a powerful and flexible platform for microbiome engineering.
Issued patent US 11,382,989