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