ABSTRACT The Lactobacillus genus is a diverse group of microorganisms, many of which are of industrial and medical relevance. Several Lactobacillus species have been used as probiotics, organisms that when present in sufficient quantities confer a health benefit to the host. A significant limitation to the mechanistic understanding of how these microbes provide health benefits to their hosts and how they can be used as therapeutic delivery systems has been the lack of genetic strategies to efficiently manipulate their genomes. This article will review the development and employment of traditional genetic tools in lactobacilli and highlight the latest methodologies that are allowing for precision genome engineering of these probiotic organisms. The application of these tools will be key in providing mechanistic insights into probiotics as well as maximizing the value of lactobacilli as either a traditional probiotic or as a platform for the delivery of therapeutic proteins. Finally, we will discuss concepts that we consider relevant for the delivery of engineered therapeutics to the human gut.