Genomic and epigenetic landscapes drive CRISPR-based genome editing in .

is a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR-based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering in subsp. demonstrating that both genomic and epigenetic contexts drive editing outcomes across strains. We reprogrammed the endogenous type I-G system to screen for naturally occurring large deletions up to 27 kb and to generate a 500-bp deletion in to abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multiple strains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly among strains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded to subsp. to emphasize the broad relevance of these findings. This study highlights the need to develop individualized CRISPR-based genome engineering approaches for distinct bacterial strains and opens avenues for engineering of next generation probiotics.