Developing the Limosilactobacillus reuteri Chassis through an Endogenous Programmable Endonuclease-Based Genome...

Using genetically tractable probiotics to engineer live biotherapeutic products (LBPs) for disease treatment is urgently needed. Limosilactobacillus reuteri is an important vertebrate gut symbiont, which has great potential for developing LBPs. However, in L. reuteri, synthetic biology work is largely limited by the long editing cycle. In this study, we identified a subtype II-A CRISPR-Cas9 system in L. reuteri 03 and found the endogenous Cas9 (LrCas9) recognizing a broad protospacer-adjacent motif (PAM) sequence (3'-NDR; N = A, G, T, C; D = A, G, T; R = A, G). We reprogrammed the LrCas9 for efficient gene deletion (95.46%), point mutation (86.36%), large fragment deletion (40 kb), and gene integration (1743 bp, 73.9%), which uncovered the function of the repeated conserved domains in mucus-binding protein. Moreover, we analyzed the distribution of endogenous endonucleases in 304 strains of L. reuteri and found the existence of programmable endonucleases in 98.36% of L. reuteri strains suggesting the potential to reprogram endogenous endonucleases for genetic manipulation in the majority of L. reuteri strains. In conclusion, this study highlights the development of a new probiotic chassis based on endogenous endonucleases in L. reuteri 03, which paves the way for the development of genome editing tools for functional genetic studies in other L. reuteri. We believe that the development of an endogenous endonuclease-based genetic tool will greatly facilitate the construction of LBPs.