Genetic Toolkit For Genome Editing of The Lactobacillus Genus.

ABSTRACTIt is now well established that the microbiome is essential to human health. Nearly every montha new study is published linking yet another disease to microbiome dysfunction. This is nolonger limited to diseases of the gastrointestinal tract and now includes multiple sclerosis,autism, asthma, Parkinson's, Alzheimer's, and cancer. Culture independent approaches such assequencing and metabolomics have provided vast amounts of information on the compositionand function of the microbiome as a whole. However, in order to enable therapeuticinterventions, further understanding of individual microorganisms, their mechanisms and theirgenes is needed. In this proposal we aim to develop a genome editing tool kit for an importantsubset of the human microbiota - the genus Lactobacillus. This tool kit will enable researchers tomodulate gene expression or abrogate gene function and allow for mechanistic studies of thefunction of specific genes as well as development of engineered microbiome therapeutics. Thephase I proposal will focus on demonstrating the feasibility and increasing the efficiency of thefirst step in the editing workflow - introduction of a chromosomal edit to a subpopulation of cellsusing ssDNA recombineering. Utilizing four Lactobacillus species in Phase I, this toolkit will giveresearchers vital access points into diverse microbiome environments in four distinctgeographies on the human body: the stomach, small intestine, large intestine and the femalereproductive tract. Phase II studies will focus on optimization of a broad host range vector forthe second step of the workflow - Cas9 facilitated selection as well as expansion to additionalLactobacillus species. Moreover the use of the tool kit will be extended to integration or deletionof larger DNA fragments providing a foundation for the development of engineered microbiometherapeutics. When commercialized this tool kit will contain vectors, electroporation reagents,media, strains and design tools that encompass the entire genome editing workflow. Thesefoundational tools will propel further microbiome discoveries and enable the development ofnovel microbiome interventions.