Portable CRISPR-Cas9 System for Flexible Genome Engineering in Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus paracasei.

Diverse strains are widely used as probiotic cultures in the dairy and dietary supplement industries, and specific strains, such as NCFM, have been engineered for the development of biotherapeutics. To expand the manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR (clustered regularly interspaced palindromic repeats)-SpyCas9 nickase (Cas9)-based system for programmable engineering of NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9 vector harboring under the regulation of a promoter and a cloning region for a customized single guide RNA (sgRNA) and editing template. The functionality of the pLbCas9 system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (, , and ), yielding 35 to 100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9 system. To showcase the versatility of this system, we also inserted an mCherry fluorescent-protein gene downstream of the gene to create a polycistronic transcript. The pLbCas9 system was further deployed in other species to generate a concurrent single-base substitution and gene deletion in ATCC 33323 and an in-frame gene deletion in Lpc-37, highlighting the portability of the system in phylogenetically distant species, where its targeting activity was not interfered with by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9 system for genome manipulations in diverse lactobacilli and open new avenues for the engineering of health-promoting lactic acid bacteria. This work describes the development of a lactobacillus CRISPR-based editing system for genome manipulations in three species belonging to the lactic acid bacteria (LAB), which are commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiotas and for their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in and further demonstrated the efficacy of this universal system in two distantly related species. This versatile Cas9-based system facilitates genome engineering compared to conventional gene replacement systems and represents a valuable gene editing modality in species that do not possess native CRISPR-Cas systems. Overall, this portable tool contributes to expanding the genome editing toolbox of LAB for studying their health-promoting mechanisms and engineering of these beneficial microbes as next-generation vaccines and designer probiotics.