Bacteriophage Vectors for in situ Microbiome Engineering

Human microbiomes are correlated with a multitude of diseases, yet remain underexploited as therapeutically, largely due to a lack of tools for performing targeted manipulations within complex microbial communities. In situ microbiome engineering using bacteriophage (phage) vectors—bacterial viruses delivering transgenic nucleic acids—represents an attractive strategy for tuning microbiomes. These vectors can be classified on their ability to replicate. Replicative phage vectors suffer from small transgene packing capacities, target cell lysis, and risks of horizontal gene transfer (HGT), whereas non-replicative phage vectors (NRPVs) have a much greater DNA packing capacity, and can avoids the risks of HGT and target cell killing. Thus, NRPVs stand as the superior platform for manipulating the microbiome. However, current NRPVs are exceedingly inefficient at transducing target bacteria, precluding their use for in situ microbiome engineering.I hypothesized the poor efficiency of NRPVs is due to the fact that dsDNA phages deliver DNA into bacteria in the linear form, which is prone to degradation and is not consistently replicated in the target bacteria as they divide. This motivated me to develop a NRPV-packable phagemid (transgenic DNA that can be loaded into a phage) system capable of circularizing itself once inside the host bacterium. I found that this system—which mediates self-circularization through a site-specific recombinase—results in potent transduction in vitro and in vivo, with >60-fold higher efficiencies than conventional NRPVs. In the process of developing these ‘self-circularizing’ NRPVs (scNRPVs), I also established a ddPCR assay for rigorous characterization of phage vectors through the use of highly transferable DNA barcodes, and I uncovered an underappreciated aspect of tRNA biology which, in turn, revealed the precise conditions mediating absolute repression of gene expression in genomically recoded, tRNA-deficient organisms. Together, these results describe a set of tools for producing and characterizing scNRPVs that have minimal risk of HGT, can deliver large transgene cassettes, and mediate exceptionally efficient transduction of target bacteria in vitro and in vivo. These tools open the possibility of performing complex, highly targeted genetic manipulations of bacteria within complex microbiomes in situ; and may be of high utility in medical, agricultural, and industrial applications.