Genetic toolkit for genome editing of the Lactobacillus genus.

ABSTRACT It is now well established that the microbiome is essential to human healthNearly every month a new study is published linking yet another disease to microbiome dysfunctionThis is no longer limited to diseases of the gastrointestinal tract and now includes multiple sclerosisautismasthmaParkinsonandapos sAlzheimerandapos sand cancerCulture independent approaches such as sequencing and metabolomics have provided vast amounts of information on the composition and function of the microbiome as a wholeHoweverin order to enable therapeutic interventionsfurther understanding of individual microorganismstheir mechanisms and their genes is neededIn this proposal we aim to develop a genome editing tool kit for an important subset of the human microbiotathe genus LactobacillusThis tool kit will enable researchers to modulate gene expression or abrogate gene function and allow for mechanistic studies of the function of specific genes as well as development of engineered microbiome therapeuticsThe phase I proposal will focus on demonstrating the feasibility and increasing the efficiency of the first step in the editing workflowintroduction of a chromosomal edit to a subpopulation of cells using ssDNA recombineeringUtilizing four Lactobacillus species in Phase Ithis toolkit will give researchers vital access points into diverse microbiome environments in four distinct geographies on the human bodythe stomachsmall intestinelarge intestine and the female reproductive tractPhase II studies will focus on optimization of a broad host range vector for the second step of the workflowCasfacilitated selection as well as expansion to additional Lactobacillus speciesMoreover the use of the tool kit will be extended to integration or deletion of larger DNA fragments providing a foundation for the development of engineered microbiome therapeuticsWhen commercialized this tool kit will contain vectorselectroporation reagentsmediastrains and design tools that encompass the entire genome editing workflowThese foundational tools will propel further microbiome discoveries and enable the development of novel microbiome interventions Microbiome dysbioses have been associated with numerous diseases including multiple sclerosisautismasthmaParkinsonandapos sAlzheimerandapos sand cancerThe genetic tools built in this proposal will enable the development of microbiome based therapeutics and help researchers gain a better understanding of how the microbiome modulates health vsdisease