Highly efficient CRISPR-Cas9 base editing in with bypass of restriction modification systems.

Intestinal microbiota members of the genus are increasingly explored as probiotics and therapeutics. However, the paucity of genetic tools and the widespread restriction modification (RM) systems in limit our ability to genetically manipulate these bacteria. Here we established a CRISPR-Cas9 cytosine base editor system (cBEST) for portable genome editing in bifidobacteria. Harboring different promoters characterized in this study, these cBEST plasmids showed a range of editing efficiencies in different strains and genomic contexts, highlighting the importance of fine-tuning base editor and sgRNA expression. Additionally, we showed that disruption or bypass of RM systems dramatically improved editing efficiencies in otherwise hard-to-edit genomic loci and strains. Notably, we demonstrated the use of RM-disrupted strains for simultaneous assembly, amplification, and methylation of the all-in-one editing plasmids, greatly streamlining the workflow for high-efficiency base editing. Last but not least, we showed the portability of cBESTs using the same editing construct to disrupt a conserved metabolic gene in multiple species. Looking ahead, the ability to efficiently edit and engineer bifidobacterial genomes will give rise to new opportunities for research and applications toward improving human health.IMPORTANCEThe ability to genetically manipulate specific genes and biological pathways in is essential to unlocking their probiotic and therapeutic potential in human health applications. The DNA double-strand break-free CRISPR-Cas9 cytosine base editor system established in this work allows portable and efficient base editing in spp. We further showed that bypass of restriction modification systems significantly improved base editing efficiency, especially for hard-to-edit genomic loci and strains. This expanded genome editing toolbox should facilitate mechanistic investigations into the roles of in host physiology and disease.